Marion

Mélanie, Céline, Daniella, Elodie, Jennifer Alice Vera

Clems, Hélène, Mériem Virginia Laure

Narjesse

Aura

Mingjun Damien Florent

Rémy Thibault

Keywords: endocytosis, 37 CIE Clathrin-independent endocytosis, 37 CIS

Remerciement

Au terme de ce travail, c'est avec émotion que je tiens à remercier tous ceux qui, de près ou de loin, ont contribué à la réalisation de ce projet.

Je tiens à remercier vivement Dr. Jacky Bonaventure et Dr. Christophe Lamaze, qui ont accepté de consacrer du temps pour évaluer mon travail en qualité de rapporteurs ainsi que Dr. Thierry Galli, Pr. Germain Trugnan et Pr. Françis Vacherot, qui ont également accepté de faire partie du jury de cette thèse.

Merci aux membres de mon comité de thèse, qui se sont rendus disponibles chaque année pour évaluer la progression de ma thèse et formuler des conseils avisés.

Un grand merci pour leur oeil d'expert à mes directeurs de thèse, Isabelle, Anh, Minh, Matt et Virginia et pour le temps consacré à la relecture et à la correction de ce manuscrit.

Mes remerciements vont également à mes deux responsables de thèse, François et Bruno, qui pendant quatre ans ont dirigé et encadré mes travaux. Merci de m'avoir ofert l'opportunité de réaliser un projet de thèse ambitieux et particulièrement de la liberté d'action que vous m'avez coniée à chaque étape de cette aventure. Aussi, merci François pour les nombreux chocolats suisses fourrés de liqueur Williams, sans oublier les oranges, pommes, clémentines, fraises à partager. Merci Bruno pour ta disponibilité lorsque je t'ai sollicité même à la dernière minute.

Je tiens également à remercier Isabelle pour ta présence lors des réunions d'avancement du projet, pour tes bons conseils ain de planiier les manipulations.

xii

List of Figures [START_REF] Aoun | Checkpoint inhibitors in bladder and renal cancers: results and perspectives[END_REF].1 Diferentially expressed genes in supericial bladder tumors from the HuExon data set 70 6.2 Diferentially expressed genes in muscle-invasive bladder tumors from the HuExon data set 71 6.3 Diferentially expressed genes in muscle-invasive bladder tumors from the TCGA data set 72 6.4 RAB25, RAB11A, RAB11B, RAB11FIP2, MYO5B expressions in bladder cancer 73 6.5 RAB25, RAB11A, RAB11B, RAB11FIP2 and MYO5B expressions in bladder cancer cell lines 74 6.6 Characteristics of bladder cancer cell lines used in the study 75 6.7 Depletion of RAB25/RAB11 and their efectors inhibits cell viability 75 6.8 Eiciency of siRNA silencing in MGHU3 and RT112 cells 76 6.9 Cell viability after depletion of RAB25/RAB11 and their efectors in several bladder cancer cell lines 77 6.10 Eiciency of siRNA silencing in bladder cancer cell lines 78 6.11 Efects on cell viability of a combination of RAB11/RAB25 depletion with FGFR3 depletion or treatment of FGFR inhibitor 80 6.12 FGFR3 preferentially localizes to the recycling compartments 81 6.13 FGFR3 accumulates in diferent compartments in cells depleted for RAB11 82 6.14 FGFR3 accumulates in diferent compartments in cells depleted for RAB11, Manders' overlap coeicients 83 6.15 Diferential efects of RAB25/RAB11 depletion on FGFR3 downstream signaling pathways 84 6.16 Expression of FGFR3 target genes after RAB25, RAB11 and/or FGFR3 depletion in RT112 cells 85 6.17 Dyngo4a treatment increases the phosphorylation of AKT and ERK1/2 but not P38 87

Introduction

Bladder cancer 1

Bladder cancer is a major health issue worldwide that causes a considerable morbidity, mortality and overall health care costs due to frequent recurrences and inefective therapeutics. It is, therefore, essential to better understand the molecular mechanisms underlying bladder tumor progression to develop targeted therapies. In this chapter, I irst introduce general facts and features about bladder cancer and then report current understanding of genetic alterations in bladder cancer.

The urinary bladder

First, I will begin by presenting some key features of the normal structure and functions of the normal bladder to more understand bladder cancer. The urinary bladder is a muscular balloon-shaped organ in the pelvic area. Its main function is to store urine from kidneys by stretching itself and to evacuates urine via ureters by contracting.

Though the bladder seems like a simple lexible sack of lesh, its structure is actually complex. The bladder wall consists of several distinct layers: the urothelium, the lamina propria, the muscularis propria and the adventitia layer. The urothelium, so-called transitional epithelium, is composed by 3 diferent cell types: basal cells, intermediate cells and surface umbrella cells (Figure 1.1). Basal cells are composed of cylindrical cells which are in contact with the basement membrane. Intermediate urothelial cells are cuboidal cells with well-deined borders. Large "umbrella" cells with an asymmetric apical membrane helps bladder relax or contract. The urothelium layer usually contacts toxic substances in urine that may explain why urothelial carcinoma is the most common type of bladder cancer, 90% [START_REF] Eble | Who classiication of ttumour. pathology and genetic of tumour of the urinary system and male genital organs[END_REF]. Lamina propria is a thin layer of connective tissue, blood vessels and nervous tissue. Muscularis propria, the thickest layer of the bladder wall, consists of spindle-shape muscle cells which provides the bladder's ability to relax and contract. Finally, the adventitia layer containing fatty connective tissue separating the bladder from adjacent organs.

Key statistics about bladder cancer

Bladder cancer has always been on the top-10 list of cancers in term of morbidity and mortality. A recent study estimated, worldwide, 429 800 new cases of bladder cancer and 165 100 deaths occurring in 2012. Europe, Northern American, Western Asia and Northern Africa have higher incidence rates than other geographic area [START_REF] Torre | Global cancer statistics[END_REF]. In Europe, an estimated 151 200 patient were newly diagnosed and 52 400 patients died from bladder cancer in 2012. In France, an estimated 11 170 new cases diagnosed and 17 150 deaths occurred in 2012 [START_REF] Ferlay | Cancer incidence and mortality patterns in europe: estimates for 40 countries in 2012[END_REF]. The American Cancer Society estimated for bladder cancer in the United States in 2015: 74 000 new cases and 16 000 deaths [START_REF] Siegel | Cancer statistics[END_REF]. Compare to other cancers, bladder cancer incidence and mortality rates have been declining or stable in developed countries over the past decades.

Bladder cancer occurs mainly in elderly people. The average age at diagnosis is seventy [START_REF] Torre | Global cancer statistics[END_REF].

The majority of bladder cancer is diagnosed in men with about a 3.8 to 1-fold difference to women in worldwide incidence rates. In men, bladder cancer is the fourth most common cancer in term of incidence and is ranked ninth leading cause of death from cancer [START_REF] Torre | Global cancer statistics[END_REF].

Tobacco smoking is the well-established risk factor of bladder cancer. It is estimated that the risk of bladder cancer in smokers is approximately 2-to 6-fold higher than in non-smokers. Exposure to toxic chemicals such as arsenic, phenols, aromatic amines increases the risk of getting bladder cancer [START_REF] Torre | Global cancer statistics[END_REF]. Growing evidences have indicated that air pollution including occupational and residential exposure to traic or traic emissions was associated with an increased risk of unitary bladder cancer [START_REF] Espina | European code against cancer 4th edition: Environment, occupation and cancer[END_REF]. Chronic infections caused by Schistosoma haematobium is mostly responsible for bladder cancer in the developing countries, particularly Africa and Western Asia [START_REF] Torre | Global cancer statistics[END_REF][START_REF] Eble | Who classiication of ttumour. pathology and genetic of tumour of the urinary system and male genital organs[END_REF].

Clinical features 5 1.3 Clinical features

Signs and symptoms. Painless gross hematuria is the most common symptom of bladder cancer (in 85% of patients) [94]. Sometimes the bleeding in the urine may be associated with pain due to the blood clotting. Tumors located at the bladder neck or having large size may cause irritative symptoms, i.e. dysuria, urgency and frequency.

Diagnosis.

Here, I present some examinations and methods proposed in the guideline for bladder cancer established by "Haute Autorité de Santé" and "Institut National du Cancer" [START_REF] Has | Guide -afection longue durée: Cancer de la vessie[END_REF].

An ultrasound examination is the irst method in case of clinical suspicion. This test allows exploring the bladder and the upper urinary systems to detect bladder cancer. Urine cytology is a systematic test which is to detect presence of abnormal cancerous cells. The test is quite speciic for cancer though it has a poor sensitivity for low grade bladder cancers. Therefore, in case of negative result, a cystoscopy will be performed. This method indicates the number of tumors, their size and their topography. The suspected lesion will be prepared for histological sections, which are examined under a microscope by a pathologist to characterize the histopathology of the tumors. When bladder cancer is detected, it is recommended to ind the localization of tumor and to evaluate the tumor by diferent imaging techniques, such as intravenous urography, magnetic resonance imaging, computational tomography.

Classiication of bladder cancer

From the diagnosis tests, the pathologists evaluate the bladder tumors according to the clinical and pathological features of tumors. First, the bladder cancers are staged using the TNM which is based on the invasive characteristic of tumor (Figure 1.2) [START_REF] Uicc | TNM classiication of Malignant Tumors 7th edition[END_REF]. Second, the tumors are graded according to their diferentiated characteristic by using the WHO 1973 or WHO/ISUP classiication 2004 [START_REF] Who | Histological typing of urinary bladder tumours[END_REF][START_REF] Eble | Who classiication of ttumour. pathology and genetic of tumour of the urinary system and male genital organs[END_REF]. The igure 1.3 summarizes the key features between these classiication systems.

At initial diagnosis, approximately 70-80% of patients with bladder cancer present non-muscle invasive papillary tumors (pTa, pT1) of low grade (G1, G2) [START_REF] Babjuk | Eau guidelines on non-muscle-invasive urothelial carcinoma of the bladder: update 2013[END_REF][START_REF] Jacobs | Bladder cancer in 2010: how far have we come?[END_REF]. These tumors frequently recur (50-70%) and less frequently progress to muscle-invasive disease (10-30%). 5-year survival is ∼ 80% [START_REF] Siegel | Cancer statistics[END_REF]. On the other hand, at initial diagnostic, 20-30% patients have muscle-invasive bladder cancer which are generally pT≥ 2 and often associated with carcinoma in situ. These tumors have poor prognosis with 5-year survival less than 40% and frequent progression to metastasis [START_REF] Siegel | Cancer statistics[END_REF].

Treatment

The actual therapeutic protocol is mainly established according to the stage and grade of the tumor described in the diagnostic assessment [START_REF] Has | Guide -afection longue durée: Cancer de la vessie[END_REF][START_REF] Babjuk | Eau guidelines on non-muscle-invasive urothelial carcinoma of the bladder: update 2013[END_REF][START_REF] Witjes | Eau guidelines on muscle-invasive and metastatic bladder cancer: summary of the 2013 guidelines[END_REF]. Other parameters The major diference is in the classiication of papillary tumors, which are classiied as grades 1, 2 and 3 in the older system and as papillary urothelial malignancy of low malignant potential (PUNLMP; equivalent to grade 1), low-grade or high-grade papillary urothelial carcinoma in the WHO/ISUP 2004 classiication [START_REF] Knowles | Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity[END_REF].

Treatment 7

such as tumor multiplicity as well as frequence of recurrences are also take into account.

Non-muscle invasive tumor. Transurethral resection (TUR) of the bladder is the irst-line treatment for non-muscle invasive tumor. Immediately after TUR, the patient can get one post-operative intravesical instillation of Mytomycin C which is an antineoplastic antibiotic inhibiting DNA synthesis. In a meta-analysis of 1476 patients, one immediate post-operative instillation had signiicant reduction of recurrence rate by 11.7% compared to TUR alone [START_REF] Sylvester | A single immediate postoperative instillation of chemotherapy decreases the risk of recurrence in patients with stage ta t1 bladder cancer: a meta-analysis of published results of randomized clinical trials[END_REF]. Additional intravesical chemotherapy instillation may be necessary depending on the risk of recurrence and of progression. Bacillus Calmette-Guerin (BCG) immunotherapy can be prescribed for the patients in case of tumors with high risk of progression. Non-muscle invasive tumors tend to recur frequently, 50-70%, and also can progress, therefore high surveillance of the urinary tract after treatment is essential.

Muscle-invasive non metastatic tumor. Radical cystectomy is the standard treatment of choice for muscle-invasive non metastatic tumors, pT2-T4a and N0M0. Radical cystectomy should be performed within 3 months after diagnosis, otherwise the risk of progression increases. Neoadjuvant and/or adjuvant chemotherapy can be used to reduce the risk of metastasis. Cisplatin-based chemotherapy will be discussed for localized bladder cancer and response to chemotherapy should be interpreted with caution. In case the patients get radiotherapy, this treatment is frequently associated with chemotherapy. It is indicated that a 10-year disease-speciic survival by 35% was better after radiotherapy and concurrent chemotherapy than after radiotherapy alone [START_REF] Witjes | Eau guidelines on muscle-invasive and metastatic bladder cancer: summary of the 2013 guidelines[END_REF].

Metastatic disease. Cisplatin-containing combination chemotherapy has been the standard irst-line systematic chemotherapy for eligible patients. Cisplatin, Methotrexate, Vinblastin, Doxorubicin regimen or Gemcitabin, Cisplatin regimen are generally used. For Cisplatin-ineligible patients, treatment with Carboplatin-containing combination chemotherapy, i.e. Gemcitabin/Carboplatin is indicated. In case tumors progress ≥6-12 months after irst-line treatment, a challenge with regimen with molecules having diferent mode of action is recommended. Bone metastases occur in 30-40% of patients with muscle-invasive and metastatic bladder cancer. Thus, Denosumad and Zoledronic acid are recommended to use [START_REF] Witjes | Eau guidelines on muscle-invasive and metastatic bladder cancer: summary of the 2013 guidelines[END_REF].

Over the two past decades, treatment has remained unchanged and the survival of bladder cancer has not been signiicantly improved with 5-year relative survival rates for regionally advanced and metastatic disease of 34% and 5%, respectively [START_REF] Siegel | Cancer statistics[END_REF]. Recently, a promising direction in bladder cancer treatment is targeted therapy by using immune checkpoint inhibitors, such as a PD-L1 antibody (in phase III trial for patients with high-risk muscle invasive bladder cancer) or PD-1 antibody (in phase II trial for patients with high-risk non-muscle invasive bladder) [START_REF] Powles | Mpdl3280a (anti-pd-l1) treatment leads to clinical activity in metastatic bladder cancer[END_REF][START_REF] Aoun | Checkpoint inhibitors in bladder and renal cancers: results and perspectives[END_REF]. Using monoclonal antibodies against FGFR3 is another promising avenue (in phase II trial for patients with locally advanced or metastatic bladder cancer, NCT02401542). A better understanding the molecular mechanisms underlying bladder tumorigenesis to help develop treatment strategies and new therapeutic targets for systematic therapy is essential.

Genetics behind bladder cancer

According to clinico-pathological features, there are two distinct groups of lesions, non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). Along with molecular proiles observed in bladder cancer, several models of two-pathway progression in bladder cancer have been proposed, Figure 1.4 represents one of them. Here, I review several alterations which are frequent events in bladder cancer. The blue and purple pathways indicate the two major pathways with distinct histopathological and molecular features that have been recognized for the past two decades. Percentages at diagnosis and key molecular features are indicated. The recent identiication of multiple distinct molecular subtypes of NMIBC and MIBC suggests multiple subpathways within each of the major pathways. It should be noted that the existence of multiple subtypes within all of the histologically recognizable intermediates remains hypothetical. Development of histopathologically recognizable urothelial alterations is preceded by clonal expansion of altered cells within the urothelium. Low-grade papillary tumours may arise via simple hyperplasia and minimal dysplasia, and these are characterized at the molecular level by loss of heterozygosity (LOH) of chromosome 9 and activating mutations of ibroblast growth factor receptor 3 (FGFR3), PIK3CA (which encodes p110 of PI3K) and stromal antigen 2 (STAG2). These non-invasive tumours recur frequently but are genetically relatively stable. Muscle -invasive carcinoma is thought to arise mainly via lat dysplasia and carcinoma in situ (CIS), which commonly show TP53 mutation in addition to chromosome 9 deletions but no FGFR3 mutations. Muscle-invasive tumours are genetically unstable and accumulate many genomic alterations. Uncertainty about the possible development of high-grade noninvasive papillary tumours from lat dysplasia is indicated by a dashed arrow. The inding of a subtype of invasive carcinomas with FGFR3 mutation and loss of cyclin-dependent kinase inhibitor 2A (CDKN2A) may suggest a route by which low-grade non-invasive papillary tumours can progress to muscle invasion (dashed arrow). ARID1A, AT-rich interactive domain 1A; EMT, epithelial-mesenchymal transition; RHOGDI1, RHO-GDP dissociation inhibitor 2; ZEB1, zinc-inger E-box binding homeobox 1 [START_REF] Knowles | Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity[END_REF].

Deletion of chromosome 9 or parts of chromosome 9 is the most frequently observed alteration in bladder cancer, in 50% of all stages and grades [START_REF] Miyao | Role of chromosome 9 in human bladder cancer[END_REF]. It is likely that deletions of chromosome 9 are present during the early stage of tumor progression [START_REF] Van Oers | Chromosome 9 deletions are more frequent than fgfr3 mutations in lat urothelial hyperplasias of the bladder[END_REF]. Several works reported that there is no diference in prevalence of chromosome 9 deletions between the 2 tumorigenesis pathways [START_REF] Hartmann | Occurrence of chromosome 9 and p53 alterations in multifocal dysplasia and carcinoma in situ of human urinary bladder[END_REF][START_REF] Pollard | Molecular genesis of nonmuscle-invasive urothelial carcinoma (nmiuc)[END_REF]. One of critical regions of deletion is 9p21 containing CDKN2A and CDKN2B, which encode for 1. [START_REF] Aoun | Checkpoint inhibitors in bladder and renal cancers: results and perspectives[END_REF] Genetics behind bladder cancer 9 the p16/p14 and p15, respectively. These genes are well known as tumor suppressor genes. Our group demonstrated that CDKN2A homozygous deletion was associated with high risk progression in non muscle-invasive tumors presenting Fibroblast growth factor receptor 3 (FGFR3) mutations [START_REF] Rebouissou | Cdkn2a homozygous deletion is associated with muscle invasion in fgfr3-mutated urothelial bladder carcinoma[END_REF].

Ta pathway. Tumors of the Ta pathway are non-invasive muscle tumors (stage pTa) which are thought to be developed via lat urothelial hyperplasia. These tumors recur frequently (50-70%) but rarely progress to stage ≥pT2 [START_REF] Jacobs | Bladder cancer in 2010: how far have we come?[END_REF]. Several well-established genetic alterations in this pathway contain FGFR3 alterations, activating alterations in Phosphatidylinositol 3-kinase (PI3K) pathway.

FGFR3 alteration is the most common event in the Ta pathway, >60%. These alterations will be discussed in more detail in section 2. [START_REF] Amornphimoltham | Rab25 regulates invasion and metastasis in head and neck cancer[END_REF].

PI3K/AKT/mTOR pathway plays an important role in cancer cell survival, proliferation [START_REF] Laplante | mtor signaling at a glance[END_REF][START_REF] Hay | Upstream and downstream of mtor[END_REF]. Figure 1.5 summarizes major actors and functions of this pathway. In tumors of the Ta pathway, PI3K pathway is frequently activated by activating mutation of the p110--catalytic subunit of PI3K (encoded by PIK3CA gene). In a recent study, 17% patients presented mutated PIK3CA in a set of 218 patients [START_REF] Sjödahl | A systematic study of gene mutations in urothelial carcinoma; inactivating mutations in tsc2 and pik3r1[END_REF]. A significant association between FGFR3 and PIK3CA mutations was also reported. Another component of this pathway, TSC1, presents loss of functions due to mutations in 17% in bladder cancer [START_REF] Sjödahl | A systematic study of gene mutations in urothelial carcinoma; inactivating mutations in tsc2 and pik3r1[END_REF]. These mutations were found to be associated with the deletion of the region 9q34 [START_REF] Knowles | Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity[END_REF].

Carcinoma in situ (CIS) pathway. Tumors of the CIS pathway are muscle-invasive high grade tumors which are thought to arise from lat urothelial dysplasia and CIS lesions. These tumors recur frequently but also progress with high probability to stage pT≥2 or metastasis. Although carcinma in situ is rare, most of the muscle-invasive tumors are thought to arise from these lesions. The major characteristic in this progression pathway is alterations in TP53, RB1 genes.

TP53 encodes for a tumor suppressor protein which is implicated in cell-cycle arrest and apoptosis. Somatic TP53 gene mutations occurring in many codons in bladder cancers have been reported in many studies [START_REF] Goebell | Bladder cancer or bladder cancers? genetically distinct malignant conditions of the urothelium[END_REF]. A recent meta-analysis of 827 tumors indicated that TP53 mutations are more frequent in high stage: pT1 (40.6%) and pT2-4 (50.7%) and also in high grade G3 (46.3%) [START_REF] Neuzillet | A meta-analysis of the relationship between fgfr3 and tp53 mutations in bladder cancer[END_REF]. In low grade, low stage tumors, FGFR3 mutations but not TP53 mutations are observed but mutations of both genes can occur in high stage-and grade-tumors of Ta pathway. Thus the authors proposed that TP53 mutations occur in the earliest stage of the CIS pathway , whereas they would occur much later in the Ta pathway, at the T1G3 or muscle-invasive stage.

RB1 encodes for a tumor suppressor protein which was identiied in retinoblastoma for the irst time. RB1 is a negative regulator of the cell cycle. Loss of heterozygosity of the RB1 locus was found in more than 50% of muscle invasive tumors at cystectomy.

The 2-pathway progression model gathered the molecular and clinicopathological features of bladder cancers that could help pathological evaluation, nonetheless, this model still does not represent the ful heterogeneity of bladder cancers. In recent years, many studies have made efort to better characterize the sub-groups of bladder cancers, especially in muscle-invasive tumors [START_REF] Sjödahl | A molecular taxonomy for urothelial carcinoma[END_REF][START_REF] Choi | Identiication of distinct basal and luminal subtypes of muscle-invasive bladder cancer with diferent sensitivities to frontline chemotherapy[END_REF][START_REF] Damrauer | Intrinsic subtypes of high-grade bladder cancer relect the hallmarks of breast cancer biology[END_REF]205,[START_REF] Biton | Independent component analysis uncovers the landscape of the bladder tumor transcriptome and reveals insights into luminal and basal subtypes[END_REF]. mTORC1 is activated by growth factors through the PI3K/AKT signaling pathway which phosphorylates and inhibits TSC1/TSC2 complex. This complex is an upstream negative regulator and acts as a GTPase-activating protein for Rheb GTPase. The activation of Rheb consequently promotes the mTOR activity which targets several downstream proteins such as 4E-BP and S6K. 4E-BP is a family of translational repressor proteins which interacts with eukaryotic translation initiation factor (eIF4E) depending their phosphorylation level. When hyperphosphorylated, 4e-BP can not bind to eIF4E which results in the recruitment of the translation initiation factor. S6K, for Ribosomal Protein S6 Kinase, regulates mRNA translation by activating or inhibiting some substrates, e.g. Ribosomal protein S6, eIF4B, eEF2K. These activities of mTOR on translational control result in the positive regulate cell growth and proliferation [START_REF] Costa-Mattioli | mtor complexes in neurodevelopmental and neuropsychiatric disorders[END_REF].

Genetics behind bladder cancer 11

A recent study from our group has allowed to deine a sub-group, named "basallike", which is characterized by an upregulation of epithelial basal cell markers including keratins (KRT5, KRT6A, KRT14) and Epidermal growth factor receptor (EGFR) and which shared a gene signature with the well-known basal subgroup in breast cancer [START_REF] Rebouissou | Egfr as a potential therapeutic target for a subset of muscle-invasive bladder cancers presenting a basal-like phenotype[END_REF]. Tumors of this sub-group are muscle-invasive and are associated with a short overall survival. EGFR pathway is activated in these tumors. Additionally, EGFR inhibitors reduce the cell proliferation and tumor appearance of cancer cells of this subgroup in vitro and in vivo. These results support therefore the advantage of classiication of sub-groups of bladder cancer according to gene expression proile for diagnostic tools and anti-cancer therapy [START_REF] Rebouissou | Egfr as a potential therapeutic target for a subset of muscle-invasive bladder cancers presenting a basal-like phenotype[END_REF].

Fibroblast growth factor receptor signaling 2

In the previous chapter, I mentioned that genetic alteration in Fibroblast Growth Factor Receptor 3 (FGFR3) is one of the most frequent events in non-muscle invasive bladder cancer. The aim of this chapter is to introduce several aspects of the Fibroblast Growth Factor receptor (FGFR) pathway, including the components, intracellular signaling cascades, physiological and pathological functions in bone development. Finally, I will discuss about FGFR3 alterations in bladder cancer.

Pathway components

2.1.1

Fibroblast Growth Factors

Fibroblast growth factor (FGF) was irst found in an extract from bovine pituitary and brain [START_REF] Gospodarowicz | Localisation of a ibroblast growth factor and its efect alone and with hydrocortisone on 3t3 cell growth[END_REF]. FGF puriied from these extracts had a mitogenic activity which stimulated the DNA synthesis of NIH-3T3 mouse embryo ibroblasts. Since this discovery, 22 members of the FGF family have been identiied and can be grouped into 7 subfamilies (Figure 2.1). 5 subfamilies of paracrine FGFs and one subfamily of endocrine FGFs mediate their cellular responses through interaction with FGF receptors. The last subfamily encodes intracellular FGFs which are not secreted and have no interaction with FGF receptors.

2.1.2

Fibroblast Growth Factor Receptors

18 secreted FGF ligands trigger their signaling by binding to diferent cell surface FGF receptors (FGFR), which are encoded by four distinct genes in mammals, FGFR1, FGFR2, FGFR3, and FGFR4 [START_REF] Ornitz | The ibroblast growth factor signaling pathway[END_REF]. FGFRs constitute a subfamily of Receptor tyrosine kinase (RTK) family. They share a common core with other RTKs with ligand-binding domains in the extracellular region, a single transmembrane helix and in a cytoplasmic region containing the protein tyrosine kinase domain and juxtamembrane regulatory region (Figure 2.2) [START_REF] Lemmon | Cell signaling by receptor tyrosine kinases[END_REF]. This receptor is composed of ∼ 800 amino acids Figure 2.1 Fibroblast Growth Factor families. Phylogenetic analysis suggests that 22 FGF genes can be classed into seven subfamilies. Branch lengths are proportional to the evolutionary distance between each gene. The FGF1, FGF4, FGF7, FGF8, and FGF9 subfamily genes encode secreted canonical FGFs, which bind to and activate FGFRs with Heparin/Heparin sulfate as a cofactor. The FGF15/19 subfamily members encode endocrine FGFs, which bind to and activate FGFRs with the Klotho family proteins ( and Klotho) as a cofactor. The FGF11 subfamily genes encode intracellular FGFs, which are non-signaling proteins serving as cofactors for voltage gated sodium channels (Nav channels) and other molecules [START_REF] Ornitz | The ibroblast growth factor signaling pathway[END_REF].

with a conserved structure comprising three extracellular Immunoglobulin (Ig)-like domains, an acidic box, a single transmembrane (TM) domain and a cytoplasmic domain containing the catalytic tyrosine kinase (TK) core (Figure 2.3.a) [START_REF] Ornitz | The ibroblast growth factor signaling pathway[END_REF]. The acidic box appears to play an important role in glycosaminoglycan modiication, and is required for modiication by heparan sulfate glycosaminoglycan. Of note, in FGFR1, 2 and 3 but not in FGFR4, FGFR isoforms are generated by alternative splicing of exons encoding their third Ig-like domain. Ig III domain is generated by splicing an invariant exon (IIIa) to either exon IIIb or IIIc, both of which splice to the exon that encodes the transmembrane region (Figure 2.3.b). In general, the IIIb of FGFR receptors are found in epithelial cells while the IIIc variants are preferentially expressed in mesenchymal cells [START_REF] Ornitz | The ibroblast growth factor signaling pathway[END_REF].

The ainity of ligand binding to the four distinct receptors is generally determined by the Ig-like domain II and III and the linker region between these 2 domains. Furthermore, in FGFR1, FGFR2 and FGFR3, the speciicity of ligand interaction is profoundly determined by alternative splicing of the FGFRb and FGFRc isoforms. The speciicity of the interaction of the 18 secreted FGFs to the diferent isoforms of FGFR has been studied by various mitogenic assay and/or by directly measuring ainity for FGFRs in BaF3 cells or L6 myoblasts that have no or little endogenous FGFR [START_REF] Ornitz | Receptor speciicity of the ibroblast growth factor family[END_REF][START_REF] Zhang | Receptor speciicity of the ibroblast growth factor family. the complete mammalian fgf family[END_REF]. Results from these studies are summarized in Figure 2.3.c. Of note, there is no ligand with high speciicity for FGFR3b apart from FGF1.

Mode of activation 17

Mode of activation

A reservoir of paracrine FGF ligands near the site of action is maintained by the interaction between FGF ligands and heparan sulfate and a speciic core proteins heparan sulfate proteoglycans which are components of extracellular matrix. To trigger the signaling, FGF is released from the extracellular matrix by heparinases, proteases or speciic FGF-binding protein. Liberated FGF subsequently binds to FGFR that leads to conformational changes and a symmetric FGF-FGFR dimer. Paracrine FGFs require heparan sulfate as co-factor to establish a stable ternary FGF-heparin-FGFR complex at a ratio 2:2:2 (Figure 2.4). In contrast, endocrine FGFs (FGF15/19 subfamily) depend on the Klotho family proteins for signaling [START_REF] Goetz | Exploring mechanisms of fgf signalling through the lens of structural biology[END_REF]. Finally, FGFR dimerization leads to transphosphorylation of the intracellular TK domain and intracellular tail that results in the activation of intracellular signaling pathways. and/or Heparan Sulfate proteoglycans (HSPG) as cofactor induces the formation of ternary FGF-heparin-FGFR complex, which activated the FGFR intracellular TK domain by phosphorylation of speciic tyrosine residues. The activated receptor is coupled to intracellular signaling pathways including the RAS-MAPK, PI3K-AKT, PLC , and STAT pathways [START_REF] Ornitz | The ibroblast growth factor signaling pathway[END_REF].

Fibroblast growth factor receptor signaling

Intracellular signaling pathways

Following ligand activation, FGFRs recruit diferent substrates to trigger signal transduction cascades or inhibitory regulators. Also, activated receptors undergo endocytosis into the cytoplasm to continue signaling or to be degraded. In this section, I will discuss about these diferent fates of activated FGFR.

2.3.1

Downstream signaling

In response to FGF-stimulation, the protein tyrosine kinase core of FGF receptors is autophosphorylated and serves as a docking site for adaptor proteins. These adaptors may be phosphorylated and then recruit other proteins to activate four major signal transductions pathways including Rat Sarcoma/Mitogen-Activated Protein Kinase (RAS/MAPK), PI3K/AKT, Phospholipase C (PLC) and Signal transducer and activator of transcription (STAT) [START_REF] Eswarakumar | Cellular signaling by ibroblast growth factor receptors[END_REF][START_REF] Turner | Fibroblast growth factor signalling: from development to cancer[END_REF][START_REF] Ornitz | The ibroblast growth factor signaling pathway[END_REF]. Figure 2.4 illustrates these four cascades of phosphorylation events.

RAS/MAPK pathway. The activation of RAS-MAPK pathway by FGF stimulation is via the recruitment of Fibroblast growth factor receptor substrate (FRS2), which speciically binds to FGFRs. The activated FGFR phosphorylates FRS2 that recruits the complex Growth Factor Receptor-Bound Protein 2 (GRB2) and son of sevenless (SOS) [START_REF] Kouhara | A lipid-anchored grb2-binding protein that links fgfreceptor activation to the ras/mapk signaling pathway[END_REF]. This complex then recruits to the membrane the inactive RAS leading to its conformational change to become active. The GTP bound active RAS subsequently recruits RAF to the plasma membrane. RAF is in turn activated and activates MEK which then phosphorylates MAPKs such as ERK1/2. Finally, the downstream targets of ERK1/2 are transcription factors including ETS, c-JUN and c-MYC [START_REF] Mccubrey | Roles of the raf/mek/erk pathway in cell growth, malignant transformation and drug resistance[END_REF]. Thus, FGFRs use the RAS/RAF/MEK/MAPK cascade to transduce signals from the cell surface to transcription factors which regulate the expression of many genes involved in proliferation, anti-apoptosis, diferentiation and cell migration.

PI3K/AKT pathway. PI3K-AKT pathway links to FGFR via the docking site FRS2-GRB2. Unlike in RAS-MAPK pathway, GRB2-associated binding protein 1 (GAB1) is the upstream activator of PI3K-AKT pathway [START_REF] Lamothe | The docking protein gab1 is an essential component of an indirect mechanism for ibroblast growth factor stimulation of the phosphatidylinositol 3-kinase/akt antiapoptotic pathway[END_REF]. GAB1 activates the enzyme PI3K leading to the phosphorylation of AKT. Activated AKT then phosphorylates Forhead box O 1 (FOXO1) which consequently results in the nuclear exclusion of FOXO1. FOXO1 is a transcription factor which has a pro-apoptotic property [START_REF] Zhang | Akt, foxo and regulation of apoptosis[END_REF].

On the other hand, phosphorylated AKT inhibits the TSC1/TSC2 complex, a modulator of mTOR pathway (Figure 1.5). In fact this complex acts as a negative regulator for Rheb GTPase. The activation of Rheb consequently promotes the mTOR activity which targets several downstream proteins such as 4E-BP and S6K. 4E-BP is a family of translational repressor proteins which interacts with eukaryotic translation initiation factor (eIF4E) depending on their phosphorylation level. When hyperphosphorylated, 4e-BP cannot bind to eIF4E, that results in the recruitment of the translation initiation factor. S6K, for Ribosomal Protein S6 Kinase, regulates mRNA translation by activating or inhibiting some substrates, e.g. Ribosomal protein S6, eIF4B, eEF2K. These activities of mTOR on translational control result in the positive regulation of cell growth and proliferation [START_REF] Hay | Upstream and downstream of mtor[END_REF].

PLC pathway. In addition to the FRS2 binding site, on the intracellular part of the activated FGFRs, another speciic phosphotyrosine at residue 766 next to the carboxy terminal can bind to PLC via the Src homology 2 domain of this protein [START_REF] Peters | Point mutation of an fgf receptor abolishes phosphatidylinositol turnover and ca2+ lux but not mitogenesis[END_REF]. Phosphorylation of PLC catalyzes Phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 serves as a second messenger that difuses in cytosol and binds to IP3 receptor on the membrane of the endoplasmic reticulum. This receptor is composed of a calcium channel at the Cterminus which helps to release calcium from the endoplasmic reticulum [START_REF] Mikoshiba | Ip3 receptor/ca2+ channel: from discovery to new signaling concepts[END_REF]. DAG is another lipid second messenger, but unlike IP3, DAG remains in the plasma membrane thanks to its hydrophobic properties. DAG activates protein kinase C (PKC) family which then phosphorylates other intracellular targets such as MAPK pathway promoting the growth of tumors [START_REF] Geraldes | Activation of protein kinase c isoforms and its impact on diabetic complications[END_REF]. Figure 2.5 illustrates the diferent components of this pathway. STAT pathway. STAT pathway is another signal transduction cascade to translate an extracellular signal into a transcriptional response.

In mammals, the STAT family consists of seven closely related proteins, including STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6. STATs are phosphorylated by activated cytokine receptors or tyrosine kinase receptors. STAT can directly bind to TK receptor but interacts to cytokine receptor via JAK. Phosphorylation of STAT at tyrosine residue results in homo-or hetero-dimeric STATs which are rapidly translocated into the nucleus. STAT dimers bind speciically to an 8-to 10-base pair inverted repeat DNA element with a consensus sequence of 5'-TT(N4-6)AA-3' within the gene promoter. Some target genes can be cited including cell-cycle inhibitor p21/WAF1, suppressors of cytokine signaling (SOCS) [START_REF] Rawlings | The jak/stat signaling pathway[END_REF]. Phosphorylated FGFR3 by ligand stimulation or mutation had been shown to activated STAT1 for the irst time in the work of Xin-Yuan Fu's group [START_REF] Su | Activation of stat1 by mutant ibroblast growth-factor receptor in thanatophoric dysplasia type ii dwarism[END_REF]. They showed that the overexpression of mutant K644E FGFR3 induced activation of STAT, leading to the nuclear translocation of the protein. Consequently, a high level of mRNA and protein of p21WAF1, a cell-cycle inhibitor was observed compared to control cells. This explains the growth arrest of 293T cells transiently expressing mutant FGFR3. STAT3 or STAT5 have been also reported to be activated by other FGFRs [START_REF] Ornitz | The ibroblast growth factor signaling pathway[END_REF].

2.3.2

Negative regulators of signaling

Following ligand stimulation, inhibition activity of receptor tyrosine kinase signaling in general and FGFR signaling in particular is essential for the precise control of cellular functions. Although the mechanism is not fully understood, several mechanisms have been proposed including degradation of the receptor, regulation of receptor kinase activity, inhibitory molecules. Some of them are represented in Figure 2.4.

Following FGF ixation on the receptor, phosphorylated FRS2 can form a ternary complex with GRB2-CBL resulting in the ubiquitination and degradation of FRS2 and FGFR. In fact, SOS and CBL compete for the same binding site (Src homology 3 domain) of GRB2 to come together with FRS2 that allows a balanced FGF signal transduction [START_REF] Wong | Frs2 alpha attenuates fgf receptor signaling by grb2-mediated recruitment of the ubiquitin ligase cbl[END_REF].

After being activated by FRS2, ERK1/2 can subsequently phosphorylate FRS2 at several threonine residues which prevents the recruitment of GRB2. This ERK-mediated negative feedback mechanism is important for the control of signaling pathways via FRS2 [START_REF] Gotoh | Regulation of growth factor signaling by frs2 family docking/scafold adaptor proteins[END_REF].

Many inhibitory regulators (molecules shaded red in igure 2.4) of receptor kinase activity have been characterized [START_REF] Ornitz | The ibroblast growth factor signaling pathway[END_REF][START_REF] Turner | Fibroblast growth factor signalling: from development to cancer[END_REF]. First, the Sprouty (SPRY) and Sproutyrelated (SPRED) proteins were identiied as an intracellular negative regulator of receptor tyrosine kinase signaling such as FGFR, EGFR, platelet-derived growth factor receptor, and nerve growth factor receptor. In FGF signaling, binding of SPRY on GRB2 prevents the recruitment of GRB2 to phosphorylated FRS2 which results in the attenuation of RAS-MAPK pathway. Of note, SPREDs are likely more potent inhibitors of MAPK activity than Sprouty [START_REF] Sivak | Fgf signal interpretation is directed by sprouty and spred proteins during mesoderm formation[END_REF]. Second, Similar Expression of FGF (SEF) is another antagonist of RAS-MAPK pathway which interacts with activated MEK then inhibits the dissociation of MEK-MAPK complex. The inhibition of SEF thus interrupts nuclear translocation of activated MAPK. SEF may act as a transmembrane protein to interact with FGFR for inhibiting receptor phosphorylation. Third, Mitogen-Activated Protein Kinase Phosphatase 3 (MKP3 or known as DUSP6) dephosphorylates ERK1/2 on phosphotyrosine and phosphothreonine residues to attenuate MAPK signaling [START_REF] Ekerot | Negativefeedback regulation of fgf signalling by dusp6/mkp-3 is driven by erk1/2 and mediated by ets factor binding to a conserved site within the dusp6/mkp-3 gene promoter[END_REF].

Intracellular signaling pathways 21

Intracellular traicking of receptor

There is now clear evidence that endocytic membrane traicking promotes receptormediated signaling and vice versa (reviewed in [START_REF] Sorkin | Endocytosis and signalling: intertwining molecular networks[END_REF][START_REF] Miaczynska | Efects of membrane traicking on signaling by receptor tyrosine kinases[END_REF]). I irst introduce the traficking of the four FGFRs and then cite two studies investigating the link between endocytic membrane traicking and signaling.

FGFR traicking. The intracellular traicking of four FGFRs was systematically studied in the work of Haugsten in 2005 for the irst time [START_REF] Haugsten | Diferent intracellular traicking of fgf1 endocytosed by the four homologous fgf receptors[END_REF]. The diferent constructions of FGFRs were introduced in HeLa cells which do not express detectable amount of any FGFR. Labeled FGF1, a ligand having a strong ainity to the four receptors, was chosen as a marker to follow the intracellular traicking of the FGFRs. After 15 minutes incubation of labeled FGF1 at 37 ∘ C, the complexes of FGF1 and diferent FGFRs were localized in Early endosome antigen 1 (EEA1)-positive early/sorting endosomes. After a 2-hour chase in the presence of leuceptin, a protease inhibitor to inhibit degradation into the lysosomes, the distribution of the four FGFRs was sorted in diferent compartments. FGFR1-3 were mainly sorted to lysosomes where it was colocalized with Lysosomal-Associated Membrane Protein 1 (LAMP1), a marker of late endosomes/lysosomes while FGFR4 showed a remarkable overlap with transferrin, a marker of recycling compartment. The biotinylation assay and radiolabelled FGF1 allowed investigating the degradation of internalized receptors and FGF1 ligand in HeLa cells expressing transiently diferent FGFRs. The results indicated that endocytosed FGFR4 was degraded more slowly than the other receptors. Additionally, internalized FGF1 was degraded more slowly in cells expressing FGFR4 than in cells expressing other FGFRs. Moreover the rate of recycling for FGF1 internalized by FGFR4 was higher than those internalized by FGFR1. FGFR4 was also shown less ubiquitinated than FGFR1 which was probably the molecular mechanism for determining the diferent sorting of the receptors after internalization [START_REF] Haugsten | Diferent intracellular traicking of fgf1 endocytosed by the four homologous fgf receptors[END_REF].

FGFR3 endocytosis.

The endocytic traicking and sorting of FGFR3 has been characterized in a few studies. The study of Haugsten and colleagues in 2011 was the irst report about the molecular mechanism involved in endocytosis of wild type FGFR3 and the role of internalization in FGFR3 signaling [START_REF] Haugsten | Clathrin-and dynamin-independent endocytosis of fgfr3implications for signalling[END_REF]. In UO2S cells expressing FGFR1 following by depletion of clathrin, the rate of labeled FGF1 internalization was signiicantly reduced compared to control cells. In contrast, in cells expressing FGFR3 knockdown for clathrin, the rate of FGF1 endocytosed was only partly reduced compared to control cells. In cells expressing FGFR3 and either wild-type dynamin or dominant negative mutant of dynamin K44A, FGF1 internalized was mostly found in EEA1 positive early endosomes. However FGF1 was excluded in EEA1 positive endosomes in FGFR1 and dynamin K44A expressing cells. Together these results showed that internalization of FGF1 via FGFR3 was partly via clathrin-and dynamindependent endocytosis while ligand-bound FGFR1 was internalized by clathrin-and dynamin-dependent manner.

To study the latter pathway of endocytosis, the authors utilized cells stably expressing FGFR3 with siRNA or dominant negative mutant of Arf6, Cdc42, Flotillin 1 and 2 which are the proteins involved in the clathrin-dependent endocytosis. The rate of internalization of labeled FGF1 via FGFR3 was then measured. The results however indicated that none of these proteins were implicated in the clathrin-and dynaminindependent endocytic pathways of FGFR3. To further investigate the impact of the internalization of FGFR3 in its signaling, the authors studied FGFR3's protein level and signaling in FGFR3-stably expressing cells that were depleted of clathrin. FGFR3 was degraded more slowly in cells depleted of clathrin but there were no remarkable changes in diferent downstream signaling pathways of FGFR3 over time. Altogether, the data from this study showed that FGFR3 used both clathrin-dependent and -independent endocytic pathways for eicient signaling [START_REF] Haugsten | Clathrin-and dynamin-independent endocytosis of fgfr3implications for signalling[END_REF].

FGFR4 intracellular traicking. The molecular mechanism underlying the recycling of FGFR4 has been recently revealed by the same authors [START_REF] Haugsten | Photoactivation approaches reveal a role for rab11 in fgfr4 recycling and signalling[END_REF]. By using biochemical assays including recycling of radiolabelled FGF1 ( 125 I-FGF1) assay and cell surface biotinylation recycling assay for FGFR4 recycling, the authors indicated that recycling of both ligand and receptor was reduced when RAB11A and RAB11B were depleted. They performed another approach providing more visualized quantitative data in which FGFR4 was fused with a photoactivatable green luorescent (PA-GFP) protein. Indeed, the traicking of photoactivated FGFR4-PA-GFP was followed at any time point of interest, for example, from endocytic recycling compartments to the plasma membrane. The results showed that this membrane transport was dependent on RAB11. To investigate more about the FGFR4-positive structure after RAB11 depletion, correlative light and electron microscopy analysis were performed. The data revealed that beyond being accumulated in tubular network, FGFR4 was localized in small vesicles budding or separating of 30-40 nm diameter. Interestingly, in RAB11depleted cells, phosphorylated FGFR4 as well as PLC was maintained for a longer period than in control cells. In contrast, the phosphorylation of AKT and ERK was found to be rapidly decreased following RAB11 knockdown [START_REF] Haugsten | Photoactivation approaches reveal a role for rab11 in fgfr4 recycling and signalling[END_REF].

Context-dependent signaling

FGF-FGFR signaling plays a fundamental role in embryogenesis, tissue homeostasis, wound healing and inlammation. At the cellular level, FGFR pathways promote different processes including proliferation, diferentiation, survival and migration. An important remark is that the functional outcome of FGF signaling depends on cellular context. In this section, I will irst introduce the physiological role of FGFR signaling in bone development. I will then report about aberrant FGF-FGFR signaling in heritable skeletal disease mutations in FGFRs focusing on FGFR3-related skeletal dysplasias.

2.4.1

FGF signaling in endochondral and intramembranous bone development

Endochondral and intramembranous bone development are two main processes contributing to form skeletal elements that occur during fetal development until late puberty. Endochondral ossiication allows the development of the skeleton, facial bones, vertebrae and the lateral medial clavicles where cartilage is replaced by bone. Intramembranous ossiication, where bone develops within sheets of connective tissue, gives rise to lat bones (e.g. the cranium and medial clavicles). Both types of ossiication need an initial condensation of mesenchyme followed by formation of calciied bone [START_REF] Ornitz | Fgf signaling pathways in endochondral and intramembranous bone development and human genetic disease[END_REF].

FGF signaling in the developing endochondral bone

The mesenchymal condensation is the irst event in the developing endochondral bone (reviewed in [START_REF] Ornitz | Fgf signaling in the developing endochondral skeleton[END_REF]). Figure 2.6 illustrates FGFR and FGF gene expression during endochondral and intramembranous bone development. FGF2, FGF9 are expressed in the developing condensation. FGFR1 is expressed in the surrounding loose mesenchyme and also in the periphery of the mesenchymal condensation. FGFR2 is however upregulated in condensing mesenchyme. How FGF signaling functions in condensing mesenchyme is however poorly understood. It is suggested that FGF signaling may regulate the expression of SOX9, an essential transcription factor for chondrocyte differentiation [START_REF] Murakami | Upregulation of the chondrogenic sox9 gene by ibroblast growth factors is mediated by the mitogen-activated protein kinase pathway[END_REF]. FGF2 and FGF9 can stimulate proliferation of chondrocytes. FGFR1 is expressed in limb mesenchyme (M) and in the periphery of the mesenchymal condensation (pink). FGFR2 is irst expressed in condensing mesenchyme (purple). FGFR3 expression is initiated as chondrocytes diferentiate and proliferate (green). FGFR1 expression is present as chondrocytes hypertrophy (red), and FGFR2 expression is prominent in osteoblasts in the ossiication center (purple). Both FGFR1 and FGFR2 appear to be coexpressed in the perichondrium (pink). (B) FGF gene expression. At the limb bud stage, FGF10 is expressed in distal mesenchyme (M) (yellow) and FGF8 is expressed in the overlying apical ectodermal ridge (AER) (pink). FGF2 and FGF9 expression is observed in the developing condensation (blue), and FGF18 expression is observed in the perichondrium and presumptive joint positions (green). FGF7 is expressed in loose mesenchyme and perichondrium surrounding the mesenchymal condensation (tan) [START_REF] Ornitz | Fgf signaling in the developing endochondral skeleton[END_REF].

Shortly after formation of the condensing mesenchyme, FGFR3 is expressed in proliferating chondrocytes in the central core of the mesenchymal condensation that suggests a direct role of FGFR3 in proliferation of chondrocytes. FGFR1 is however expressed in prehypertrophic and hypertrophic chondrocytes which suggests a role for FGFR1 in controlling cell survival, cell diferentiation and cell death. FGFR2 continues to be expressed in osteoblasts in the ossiicated region of mature bone.

Endochondral bone development requires an orchestrated regulation between chondrocyte proliferation, diferentiation to hypertrophic chondrocyte and ossiication. These signaling pathways will be more discussed in the section 2.4.2.2.

FGF signaling in the developing intramembranous bone

Intramembranous ossiication is responsible for the formation of the cranium and medial clavicles. Intramembranous ossiication begins with the mesenchymal condensation. Undiferentiated mesenchymal cells then diferentiate into osteoprogenitor cells which become bone matrix-forming mature osteoblasts. Osteoblasts either die by apoptosis or are embedded in the matrix, becoming osteocytes [START_REF] Ornitz | Fgf signaling pathways in endochondral and intramembranous bone development and human genetic disease[END_REF].

During this progressive process, the expression of FGFs and FGFRs is temporally and spatially controlled to assure the balance between proliferation, apoptosis and differentiation of osteogenic cells. FGF2, FGF18, FGFR1 and FGFR2 are the key regulators in intramembranous bone formation [START_REF] Ornitz | Fgf signaling pathways in endochondral and intramembranous bone development and human genetic disease[END_REF]. In recent years, essential molecular mecha- nisms by which FGF/FGFR signaling promotes intramembranous bone development has been revealed by analyzing FGFR mutations involved in human skeletal disorders and using genetically altered mouse models. Three downstream pathways in response to FGF-stimulation are well characterized during osteoblastogenesis. There are ERKs, AKT and PKC pathways which are coordinated to promote cell proliferation and differentiation (Figure 2.7). First, activation of ERK1/2 signaling is partly involved in cell proliferation of osteoprogenitors [START_REF] Choi | Fibroblast growth factor-2 and -4 promote the proliferation of bone marrow mesenchymal stem cells by the activation of the pi3k-akt and erk1/2 signaling pathways[END_REF]. Osteoblast diferentiation promoted by FGFR2 is thought via ERK-induced expression of RUNX2, an essential transcription factor involved in osteoblastogenesis. The activation of ERK-MAP kinase also induces expression of osteogenic marker genes [START_REF] Park | Functional characterization of a novel fgfr2 mutation, e731k, in craniosynostosis[END_REF]. Second, PKC activation by FGFR2 also results in RUNX2 activity in mesenchymal cells and osteoblasts [START_REF] Miraoui | Fibroblast growth factor receptor 2 promotes osteogenic diferentiation in mesenchymal cells via erk1/2 and protein kinase c signaling[END_REF]. Third, PI3K-AKT pathway is activated by FGF2 or FGF4 that promotes the proliferation and diferentiation

Context-dependent signaling 25

of osteoblast precursor cells [START_REF] Choi | Fibroblast growth factor-2 and -4 promote the proliferation of bone marrow mesenchymal stem cells by the activation of the pi3k-akt and erk1/2 signaling pathways[END_REF].

Many studies also demonstrated that there are crosstalks between FGF/FGFR signaling, Wnt signaling and other receptor tyrosine kinase signaling which control osteoblast functions (further detail in [START_REF] Marie | Fibroblast growth factor signaling controlling bone formation: an update[END_REF]).

2.4.2

Heritable skeletal disease mutations in FGFRs

Due to the essential role of FGFR1, 2 and 3 in skeletal development, alterations in particular the mutations of these receptors, are the etiology in many heritable skeletal diseases.

Craniosynostosis

Craniosynostosis consists of premature fusion of one or more cranial sutures, occuring in ∼1 in 2500 newborns [94]. Syndromic craniosynostosis is usually associated with many complications afecting sensory, respiratory and neurological function. The majority of craniosynostosis syndromes are associated with mutations in FGFR2. Autosomal dominant gain-of-function missense mutations, deletions, and insertions in FGFR2 result in Apert syndrome, Crouzon syndrome, non syndromic craniosynostosis syndrome, Saethe-Chotzen syndrome, Pfeifer syndrome, and Jackson-Weiss syndrome [94,[START_REF] Ornitz | The ibroblast growth factor signaling pathway[END_REF]. The consequence of the classic Apert syndrome mutations (S252W and P253R FGFR2) is modiication of ligand binding ainity. Gain-of-function missense mutations in FGFR1 are found in several craniosynostosis syndromes including Pfeifer syndrome, Jackson-Weiss syndrome, Muenke syndrome, and osteoglophonic dysplasia. Several mutations of FGFR3 can cause speciic craniosynostosis syndromes but at low prevalence (about 5-10%) [94].

Chondrodysplasia syndromes

Chondrodysplasia syndromes are characterized by reduced growth of long bones with proximal elements more severely afected than distal elements. Since the point mutation in FGFR3 in achondroplasia was irst reported in 1994 [START_REF] Shiang | Mutations in the transmembrane domain of fgfr3 cause the most common genetic form of dwarism, achondroplasia[END_REF][START_REF] Rousseau | Mutations in the gene encoding ibroblast growth factor receptor-3 in achondroplasia[END_REF], many studies have contributed to identify diferent mutations in FGFR3 in the dwarism chondrodysplasia syndromes, which include hypochondroplasia (HCH), achondroplasia (ACH), severe ACH with developmental delay and acanthosis nigricans (SADDAN), thanatophoric dysplasia (TD) (TDI and II). ACH is the most common form of shortlimb dwarisms, and its birth incidence is estimated to occur in 1 in 10000 to 1 in 30000 newborns [87]. The consequences of FGFR3 mutations in these syndromes will be discussed further below.

Mutations of FGFR3 in diferent forms of chondrodysplasia syndromes

Like others FGF receptors, FGFR3 has three extracellular Ig-like domains (I, II, III), a single transmembrane domain and an intracellular domain that contains a split tyrosine kinase subdomain. The alternative splicing of the third Ig-like domain produces 2 isoforms of FGFR3 (FGFR3b and FGFR3c). FGFR3b is mainly expressed in epithelial cell populations while the form FGFR3c is present in non-epithelial cells. Among 22 known FGFs, the speciic physiological ligand(s) for FGFR3 is still not identiied, although FGFs 1, 2, 4, 9, and 18 are probably the best candidates based on the distribution of expression, the capacity of binding and activating FGFR3, and the phenotype of mice lacking these particular ligands [START_REF] Ornitz | Fgf signaling in the developing endochondral skeleton[END_REF][START_REF] Zhang | Receptor speciicity of the ibroblast growth factor family. the complete mammalian fgf family[END_REF][START_REF] Liu | Coordination of chondrogenesis and osteogenesis by ibroblast growth factor 18[END_REF]. From the irst discovery of FGFR3 mutation in skeletal dysplasias, 18 mutations have been identiied. FGFR3 mutations in skeletal dysplasias are summarized in igure 2.8.

The common consequence of these mutations is constitutive FGFR3 activation. The transmembrane mutation (G380R in ACH) is thought to ligand-independent stabilization of FGFR3 dimers via hydrogen bonds formed between the side-chains of the two arginine residues [START_REF] Webster | Constitutive activation of ibroblast growth factor receptor 3 by the transmembrane domain point mutation found in achondroplasia[END_REF]. TDI-associated Y373C and R248C substitutions activate FGFR3 via forming covalently bound dimers by a disulide bond between the free cysteine residues which are introduced into the juxtamembrane domain (Y373C) or to the region linking two Ig domains in the extracellular part of FGFR3 (R248C) [START_REF] Robertson | Constitutive activation of ibroblast growth factor receptor 3 by mutations responsible for the lethal skeletal dysplasia thanatophoric dysplasia type i[END_REF][START_REF] Naski | Graded activation of ibroblast growth factor receptor 3 by mutations causing achondroplasia and thanatophoric dysplasia[END_REF]. Finally, mutations in the activation loop kinase domain, such as K650M (TDI) or K650E (TDII), result in constitutive FGFR3 activation by mimicking the conformational shifts in this domain that are normally induced by ligand-stimulation followed by autophosphorylation [START_REF] Webster | Constitutive activation of ibroblast growth factor receptor 3 by the transmembrane domain point mutation found in achondroplasia[END_REF].

Lievens and colleagues proposed that diferent types of amino acid substitution at the Lysine 650 of FGFR3 interferes with its biosynthesis and its intracellular transport.

Two highly phosphorylated FGFR3 (K650M and K650E), which are associated with severe pathologies, were located as immature glycosylated proteins in the endoplasmic reticulum (ER), and failed to be degraded. They could even activate JAK/STAT signaling pathway from ER [START_REF] Lievens | The thanatophoric dysplasia type ii mutation hampers complete maturation of ibroblast growth factor receptor 3 (fgfr3), which activates signal transducer and activator of transcription 1 (stat1) from the endoplasmic reticulum[END_REF][START_REF] Lievens | The kinase activity of ibroblast growth factor receptor 3 with activation loop mutations afects receptor traicking and signaling[END_REF]. The ER localization of murine K644E FGFR3 corresponding to the human K650E substitution in ER is still controversial, because these results in HEK293 cells were diferent compared to the same construction in COS7 cells where mutant FGFR3 was located in vesicular structures [START_REF] Cho | Defective lysosomal targeting of activated ibroblast growth factor receptor 3 in achondroplasia[END_REF].

Another consequence of mutant FGFR3 is the increased stability of the receptor. The work of Cho and colleagues suggested that ACH-G380R and TDII-K650E disrupted c-Cbl-mediated ubiquitination that resulted in the escape of mutant receptor from lysosomal targeting into recycling pathway to increase the signaling capacity [START_REF] Cho | Defective lysosomal targeting of activated ibroblast growth factor receptor 3 in achondroplasia[END_REF].

Although all FGFR3-related skeletal dysplasias are characterized by shortening of the long bones, they display a graded spectrum of phenotypic severity, ranging from relatively mild HCH to the neonatal lethal TD. Some studies have suggested that the phenotypic severity depends on the phosphorylated states, the intracellular localization of the mutated FGFR3 and the duration of FGFR3 phosphorylation [START_REF] Naski | Graded activation of ibroblast growth factor receptor 3 by mutations causing achondroplasia and thanatophoric dysplasia[END_REF][START_REF] Harada | Sustained phosphorylation of mutated fgfr3 is a crucial feature of genetic dwarism and induces apoptosis in the atdc5 chondrogenic cell line via plcgamma-activated stat1[END_REF]. Figure 2.9 shows the factors which potentially decide the severity of diferent FGFR3-related chondrodysplasia syndromes.

FGFR3 signaling in chondrodysplasia syndromes

FGFR3 functions as a physiological negative regulator of growth of long bones. FGFR3 signaling plays diferent roles in chondrocyte including inhibiting the proliferation, promoting the diferentiation, regulating the synthesis of chondrocyte extracellular matrix and cellular shape. The dwarism phenotypes in patients carrying gain-ofmutations in FGFR3 are thought to exaggerate the physiological function [START_REF] Foldynova-Trantirkova | Sixteen years and counting: the current understanding of ibroblast growth factor receptor 3 (fgfr3) signaling in skeletal dysplasias[END_REF].

Three main signaling pathways of FGFR3 implicating chondrodysplasia syndromes are: STAT, ERK-MAPK, stabilization of FGFR3. Figure 2.10 illustrates these pathways. alters chondrocyte behavior by inducing premature growth arrest, loss of extracellular matrix, altered differentiation, and changes in cell shape. Together, these cellular phenotypes (gray arrows) contribute to profound disruption of the growth plate cartilage resulting in skeletal dysplasia. At the molecular level, the growth arrest phenotype is mediated by induction of several inhibitors of the cell cycle, belonging to cip/kip family (p21) or INK4 family (p16, p18, p19), whereas the loss of the extracellular matrix originates from both inhibition of production of major matrix components (collagen type II and aggrecan), and active matrix degradation, mediated by several members of MMP family. Expression of two important physiological regulators of chondrocyte diferentiation, Indian hedgehog (Ihh) and parathyroid hormone-related protein (PTHrP), is inhibited by FGFR3 in cartilage, likely contributing to impaired chondrocyte hypertrophic diferentiation. ERK MAP kinase is a major pathway for growth arrest, extracellular matrix loss, and impaired chondrocyte diferentiation. FGFR3 causes prolonged activation of the ERK signaling module (RAS-RAF-MEK-ERK), mediated by adapter (GAB1, FRS2, and SHC) driven recruitment of SHP2-GRB2-SOS1 complexes to the cell membrane, where they activate RAS. In addition, SNAIL1 transcription factor is involved in regulation of FGFR3-mediated ERK activity, although the exact nature of this regulation is not presently clear (question marks). The FGFR3-mediated activation of the ERK pathway is antagonized by CNP signaling, which inhibits ERK pathway by inactivation of RAF kinase, via inhibitory phosphorylation mediated by cGMP-activated protein kinase (PKG). Some FGFR3 mutants also activate STAT1, possibly via direct phosphorylation at Y701. It is, however, currently unclear whether activated STAT1 or other STATs induce cell cycle inhibitor expression in cartilage, thereby contributing to the FGFR3-mediated growth arrest (question mark). Finally, chronic activation of FGFR3 leads to inhibition of canonical cytokine-STAT signaling in chondrocytes, via both induction of SOCS inhibitors of cytokine signaling and inhibition of expression of receptors for IL6 (IL6R ) or LIF (LIFR). As the latter cytokines represent positive regulators of chondrocyte proliferation, their inhibition might contribute to the pathological efects of FGFR3. NPR-B, natriuretic peptide receptor B/guanylyl cyclase B; GTP, guanosine-5'-triphosphate; cGMP, cyclic guanosine monophosphate; gp130, glycoprotein 130 [START_REF] Foldynova-Trantirkova | Sixteen years and counting: the current understanding of ibroblast growth factor receptor 3 (fgfr3) signaling in skeletal dysplasias[END_REF].

Context-dependent signaling 29

Signal Transducer and Activator of Transcription (STAT) is characterized as a mediator in mutated FGFR3 signaling in many studies. STAT signal was irst reported in Su's work [START_REF] Su | Activation of stat1 by mutant ibroblast growth-factor receptor in thanatophoric dysplasia type ii dwarism[END_REF]. They showed that mutant TDII FGFR3 (K650E) can speciically induce activation and nuclear translocation of the transcription factor STAT1. This correlates with the accumulation in the nuclei of p21(WAF/CIF), a cell-cycle inhibitor, and growth arrest of the 293T cells expressing K650E FGFR3. It was suggested that STAT1 activation and P21 induction might be responsible for delayed growth in bone development. Many studies have then given evidences to support this model in which lethal FGFR3 mutants (K650M or K650E) use STAT1 as a mediator to regulate chondrocyte proliferation and diferentiation [START_REF] Sahni | Fgf signaling inhibits chondrocyte proliferation and regulates bone development through the stat-1 pathway[END_REF][START_REF] Murakami | Constitutive activation of mek1 in chondrocytes causes stat1-independent achondroplasia-like dwarism and rescues the fgfr3-deicient mouse phenotype[END_REF][START_REF] Lievens | The kinase activity of ibroblast growth factor receptor 3 with activation loop mutations afects receptor traicking and signaling[END_REF][START_REF] Legeai-Mallet | Overexpression of fgfr3, stat1, stat5 and p21cip1 correlates with phenotypic severity and defective chondrocyte diferentiation in fgfr3-related chondrodysplasias[END_REF][START_REF] Harada | Sustained phosphorylation of mutated fgfr3 is a crucial feature of genetic dwarism and induces apoptosis in the atdc5 chondrogenic cell line via plcgamma-activated stat1[END_REF]. Strikingly, Krejci and his colleagues systematically compared the capacity of phosphorylation by STATs of wild type FGFR3 and diferent mutants which are associated with chondrodysplasia syndromes. Only K650M and K650E FGFR3 mutants of tyrosine kinase domain, signiicantly activated STAT1 or STAT5 [START_REF] Krejci | Stat1 and stat3 do not participate in fgf-mediated growth arrest in chondrocytes[END_REF]. These results were similar to data from other studies in which K650 mutants were used as models to identify the pathways implicated in bone growth in FGFR3-related skeletal dysplasia. Krejci and his colleagues suggested that STAT pathway is just an unusual signaling restricted to K650 Mutants (reviewed in [START_REF] Foldynova-Trantirkova | Sixteen years and counting: the current understanding of ibroblast growth factor receptor 3 (fgfr3) signaling in skeletal dysplasias[END_REF]). They proposed an alternative model for the implication of STATs in FGFR3 downstream signaling pathways that FGFR3 may inhibit directly canonical cytokine-STAT signaling activated by interferon , Interleukin 6, Interleukin 11, and leukemia inhibitory factor (LIF); or indirectly via induction of Suppressor Of Cytokine Signaling 1 and 3 (SOCS1 and SOCS3), inhibitors of cytokine signaling. Thus, constitutively activated FGFR3 in FGFR3-related skeletal dysplasia might inhibit cytokine-STAT signaling leading to altered chondrocyte proliferation and diferentiation (Figure 2.10).

Many studies have revealed that the RAS-RAF-MEK-ERK pathway is activated by mutated FGFR3 in ACH and TD. In Murakami's work, mice expressing the achondroplasia mutant of FGFR3 displayed uncompleted hypertrophic chondrocytes and a high phosphorylation of MEK1 in the growth plate. In addition, expression of a constitutively active mutant of MEK1 in chondrocytes of fgfr3-/-mice delayed hypertrophic chondrocyte diferentiation. These results suggested a model in which FGFR3 signaling uses the MAPK pathway to inhibit chondrocyte diferentiation [START_REF] Murakami | Constitutive activation of mek1 in chondrocytes causes stat1-independent achondroplasia-like dwarism and rescues the fgfr3-deicient mouse phenotype[END_REF]. Lievens and colleagues demonstrated that mice K644E/M mutants, corresponding to K650E/M in human, which causes the severe skeletal dysplasia SADDAN and TDII, may activate ERK1/2 from the endoplasmic reticulum [START_REF] Lievens | K644e/m fgfr3 mutants activate erk1/2 from the endoplasmic reticulum through frs2 alpha and plc gammaindependent pathways[END_REF]. Moreover, constitutive activation of FGFR3 inhibited longitudinal bone growth by decreasing the extracellular matrix synthesis through the MAPK pathway [START_REF] Yasoda | Overexpression of cnp in chondrocytes rescues achondroplasia through a mapk-dependent pathway[END_REF].

In ACH or TD growth plates, FGFR3 mutation has a tendency to be upregulated in comparison to wild type FGFR3. It may be due to the fact that mutated FGFR3 escapes from lysosomal degradation by c-CBL-dependent ubiquitination, that results in recycling of activated receptors and ampliication of FGFR3 signals [START_REF] Cho | Defective lysosomal targeting of activated ibroblast growth factor receptor 3 in achondroplasia[END_REF]. Results from the same group [START_REF] Guo | Sprouty 2 disturbs fgfr3 degradation in thanatophoric dysplasia type ii: a severe form of human achondroplasia[END_REF] further proposed a precise mechanism of this model: constitutively active FGFR3 induces phosphorylation of SPRY2 leading to increased c-CBL sequestration by SPRY2. These results are consistent with previous observations that c-CBL is unable to interact with mutant FGFR3 for ubiquitination and degradation. However, the implication of c-CBL in degradation of mutated FGFR3 by ubiquitination is still controversial, as those of data mentioned above are diferent from other studies reporting strong ubiquitination of other FGFR3 mutants having no efect on FGFR3 stability [START_REF] Monsonego-Ornan | Fgf receptors ubiquitylation: dependence on tyrosine kinase activity and role in downregulation[END_REF][START_REF] Bonaventure | The localization of fgfr3 mutations causing thanatophoric dysplasia type i diferentially afects phosphorylation, processing and ubiquitylation of the receptor[END_REF].

2.4.3

Deregulation of FGFR signaling in cancer

Deregulation of FGFR signaling pathways can be originated from aberrant expression, mutation in FGF ligands and receptors, or from alterations in downstream signaling pathways. This deregulation has been found in many types of cancers (extensively reviewed in [START_REF] Turner | Fibroblast growth factor signalling: from development to cancer[END_REF]). Figure 2.11 summarizes the contribution of FGF ligands and FGF receptors in cancer. Particularly, in bladder cancer, FGFR3 alteration is one of the most frequent events. Thus, I dedicate the next section 2.5 to introduce the alterations of FGFR3 in bladder cancer.

Alterations of FGFR3 in bladder cancer

As mentioned above, the functions of FGFR signaling in general and FGFR3 signaling in particular depend on the cellular context. FGFR3 acts as a physiological negative regulator of skeletal growth via inhibition of chondrocyte proliferation and the gain-of-function mutations of FGFR3 cause the enhanced inhibition in this cell type. However, the gain-of-function mutations of FGFR3, even the same point mutations, cause excessive cell proliferation in epithelial cells [START_REF] Logié | Activating mutations of the tyrosine kinase receptor fgfr3 are associated with benign skin tumors in mice and humans[END_REF][START_REF] Bernard-Pierrot | Oncogenic properties of the mutated forms of ibroblast growth factor receptor 3b[END_REF]. In this section, I will report the current understanding of the alterations of FGFR3 and their consequences in tumorigenesis and signaling in bladder cancer.

2.5.1

Activating mutations

FGFR3 mutations in bladder cancer were irst reported by Radvanyi's group over ifteen years ago [START_REF] Cappellen | Frequent activating mutations of fgfr3 in human bladder and cervix carcinomas[END_REF]. They found single nucleotide substitutions of FGFR3 in 9 of 26 bladder carcinomas (35%). Since then, 11 diferent mutations have been identiied in urothelial tumors. All FGFR3 mutations described to date are shown in Figure 2.12. The most frequent mutations are S249C (66.6%), Y375C (15.1%), R248C (9.7%) and G372C (4.3%). They are identical to the mutations causing lethal TDI (Figure 2.8). Data from our group and cBioPortal indicate that FGFR3 mutations seem to be restricted to a few tumor types including bladder cancer (50-60% in non-invasive muscle tumors, 12-20% in muscle-invasive tumors), lung cancer (around 6%), melanoma (around 3%). Other types of cancers have low frequency of FGFR3 mutations [START_REF] Karoui | No evidence of somatic fgfr3 mutation in various types of carcinoma[END_REF][START_REF] Gao | Integrative analysis of complex cancer genomics and clinical proiles using the cbioportal[END_REF][START_REF] Cerami | The cbio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data[END_REF].

Many studies established that FGFR3 mutations is strongly associated with low tumor grade and stage [START_REF] Kimura | The incidence of thanatophoric dysplasia mutations in fgfr3 gene is higher in low-grade or supericial bladder carcinomas[END_REF][START_REF] Lindgren | Molecular characterization of early-stage bladder carcinomas by expression proiles, fgfr3 mutation status, and loss of 9q[END_REF][START_REF] Tomlinson | Fgfr3 protein expression and its relationship to mutation status and prognostic variables in bladder cancer[END_REF][START_REF] Neuzillet | A meta-analysis of the relationship between fgfr3 and tp53 mutations in bladder cancer[END_REF]. The recent meta-analysis of 916 tumors about the distribution of FGFR3 mutations by stage and grade indicated that the frequency of FGFR3 mutations signiicantly decreased while gravity of stage and grade increased. In term of stage, 65% pTa tumor samples, 30% of pT1 tumor samples, 11.5% in pT2-4 tumors samples present mutant FGFR3. Also, by grade, 69.8% G1 tumors, 68% G2 tumors and only 18.6% of total G3 tumor sample present mutant FGFR3 [START_REF] Neuzillet | Fgfr3 mutations, but not fgfr3 expression and fgfr3 copy-number variations, are associated with favourable non-muscle invasive bladder cancer[END_REF]. The S249C or Y375C mutations create a cysteine residue in the extracellular domain or the transmembrane region of the receptor, respectively that results in the formation of disulide bonds between adjacent monomer receptors [START_REF] Robertson | Constitutive activation of ibroblast growth factor receptor 3 by mutations responsible for the lethal skeletal dysplasia thanatophoric dysplasia type i[END_REF]. This ligandindependent dimerization leads to the constitutive activation of mutant receptors [START_REF] Bernard-Pierrot | Oncogenic properties of the mutated forms of ibroblast growth factor receptor 3b[END_REF]. Mutations in kinase domain (lysine residue at position 652, K650E, identical to the TDII mutation) which are more frequent in non-lethal dysplasias, are rare in urothelial cancer with frequency of around 2%. In skeletal dysplasia, these mutations are thought to mimic the conformational changes leading to ligand-independent receptor activation and signaling [START_REF] Webster | Constitutive activation of ibroblast growth factor receptor 3 by the transmembrane domain point mutation found in achondroplasia[END_REF].

2.5.2

Overexpression

The levels of FGFR3 transcripts and protein examined by RT-qPCR or immunohistochemistry, respectively have been reported in several studies [START_REF] Gómez-Román | Fibroblast growth factor receptor 3 is overexpressed in urinary tract carcinomas and modulates the neoplastic cell growth[END_REF][START_REF] Bernard-Pierrot | Oncogenic properties of the mutated forms of ibroblast growth factor receptor 3b[END_REF][START_REF] Tomlinson | Fgfr3 protein expression and its relationship to mutation status and prognostic variables in bladder cancer[END_REF][START_REF] Bodoor | Fgfr3 mutational status and protein expression in patients with bladder cancer in a jordanian population[END_REF]. There was an association of FGFR3 expression levels with tumor stage and grade. Overexpression of FGFR3 was more prevalent in low-stage and low-grade tumors. The expression of FGFR3 in pTa and pT1 tumors was increased compared to normal human urothelial cells. Moreover, supericial bladder tumors expressed signiicantly higher FGFR3 than muscle-invasive tumors [START_REF] Bernard-Pierrot | Oncogenic properties of the mutated forms of ibroblast growth factor receptor 3b[END_REF][START_REF] Tomlinson | Fgfr3 protein expression and its relationship to mutation status and prognostic variables in bladder cancer[END_REF]. Interestingly, there was a correlation between mutation status and expression level of FGFR3. Mutant tumors had increased FGFR3 mRNA levels by 2.2-and 3.1-fold compared to normal human urothelial cells and non-mutated tumors, respectively [START_REF] Bernard-Pierrot | Oncogenic properties of the mutated forms of ibroblast growth factor receptor 3b[END_REF]. A recent study by analyzing tissue mi-

Alterations of FGFR3 in bladder cancer 33

croarrays has been in accordance with this observation [START_REF] Poyet | Positive ibroblast growth factor receptor 3 immunoreactivity is associated with low-grade non-invasive urothelial bladder cancer[END_REF]. They have shown that FGFR3 was identiied in 113/207 tumor samples (54.6%) and was signiicantly associated with FGFR3 mutation, low tumor stage, low histological grade and a papillary growth pattern. 74% of tumors with positive FGFR3 expression presented an FGFR3 mutation.

The molecular mechanisms driving the overexpression of FGFR3 in bladder cancer however are partly understood. One study of microRNA (miR) alterations in 78 normal and malignant urothelial samples indicated that loss of miR-99a and miR-100 leads to overexpression of FGFR3 in tumors [START_REF] Catto | Distinct microrna alterations characterize high-and low-grade bladder cancer[END_REF]. Normally, these microRNAs bind directly to the 3'UTR region of the FGFR3 mRNA leading to the degradation of the mRNA.

One group found overexpression of FGFR3 in 50% of muscle-invasive tumors in which wild type FGFR3 was expressed more commonly [START_REF] Gómez-Román | Fibroblast growth factor receptor 3 is overexpressed in urinary tract carcinomas and modulates the neoplastic cell growth[END_REF]. Thus, it is suggested that FGFR3 is necessary for bladder tumor progression in both supericial and muscleinvasive tumors.

2.5.3

Translocations

Chromosomal translocation is another mechanism of abnormal FGFR3 activation in bladder cancer. Figure 2.13 shows two types of FGFR3 fusion identiied in bladder cancer, FGFR3-TACC3 and FGFR3-BAIAP2L1. The fusion gene which occurs between FGFR3 and the transforming acidic coiledcoil containing protein 3 (TACC3) gene has been reported in glioblastoma in 2012 for the irst time [START_REF] Singh | Transforming fusions of fgfr and tacc genes in human glioblastoma[END_REF]. They found that the fusion gene has oncogenic properties. Indeed, cells expressing FGFR3-TACC3 was able to grow in anchorage-independent conditions and the mice transduced with FGFR3-TACC3 developed malignant brain tumors. FGFR3-TACC3 displayed constitutive phosphorylation and inhibition of FGFR kinase rescued the aneuploidy defect and oral administration of an FGFR inhibitor prolongs survival of mice bearing intracranial FGFR3-TACC3-initiated glioma. FGFR3-TACC3 has been subsequently identiied in other cancers including lung cancer, head and neck cancer, oral cancer, cervical cancer and also bladder cancer [START_REF] Capelletti | Identiication of recurrent fgfr3-tacc3 fusion oncogenes from lung adenocarcinoma[END_REF][START_REF] Yuan | Recurrent fgfr3-tacc3 fusion gene in nasopharyngeal carcinoma[END_REF][START_REF] Carneiro | Fgfr3-tacc3: A novel gene fusion in cervical cancer[END_REF][START_REF] Williams | Oncogenic fgfr3 gene fusions in bladder cancer[END_REF].

In bladder cancer, to date, several isoforms of FGFR3-TACC3 have been identiied. The group of Knowles found 3 isoforms which contain commonly exon 1-18 FGFR3 fused with diferent lengths of TACC3: from exon 11 in RT112 or LUCC2 cell lines, from exon 4 in RT4 cell line, and from exon 8 in one tumor sample [START_REF] Williams | Oncogenic fgfr3 gene fusions in bladder cancer[END_REF]. TCGA study identiied 2 isoforms of FGFR3-TACC3 of which the breakpoint was in intro 16 or exon 17 of FGFR3 and intro 10 of TACC3 [205]. Results from our laboratory by Western Blot and PCR indicated that FGFR3-TACC3 were detected in 4 of 200 tumor samples examined. Chromosomal rearrangement results in the loss of the 3'-UTR of FGFR, a region that contains various microRNA regulation sites. In glioblastoma, it was shown that the formation of FGFR3-TACC3 fusion resulted in a loss of microRNA-99a site leading to the overexpression of FGFR3-TACC3 [START_REF] Parker | The tumorigenic fgfr3-tacc3 gene fusion escapes mir-99a regulation in glioblastoma[END_REF]. This could also explain why FGFR3-TACC3 is generally overexpressed in bladder cancer cell lines or tumor samples.

FGFR3-BAI1-associated protein 2-like 1 (BAIAP2L1) is another gene fusion identiied in bladder cancer. FGFR3-BAIAP2L1 has been detected in 4 of 46 patients with bladder cancer [START_REF] Nakanishi | Mechanism of oncogenic signal activation by the novel fusion kinase fgfr3-baiap2l1[END_REF] and in one bladder cancer cell line, SW780 [START_REF] Williams | Oncogenic fgfr3 gene fusions in bladder cancer[END_REF].

2.5.4

Consequences in the downstream signaling of FGFR3 alterations

It has been shown that FGFR3 mutants display oncogenic properties in bladder cancers. Indeed, they have transforming capacity in vitro and induce the tumorigenicity in vivo mouse models. NIH-3T3 cells expressing S249C FGFR3 increases cell proliferation and presents anchorage-independent cell growth. In addition, in nude mice xenografted with cells expressing S249C FGFR3, tumors develops after 10 days [START_REF] Bernard-Pierrot | Oncogenic properties of the mutated forms of ibroblast growth factor receptor 3b[END_REF]. In the same study, the authors indicated that Y375C mutation is required for the transforming properties of MGH-U3 cell line. Another study conirms these observations in NIH-3T3 cells, however, in immortalized normal human urothelial TERT-NHU cells, S249C and Y375C mutants promotes cell proliferation and viability [START_REF] Di Martino | Mutant ibroblast growth factor receptor 3 induces intracellular signaling and cellular transformation in a cell type-and mutation-speciic manner[END_REF]. Also, the knockdown of S249C FGFR3 in the urothelial cell line 97-7 reduces the proliferation and anchorage independent growth [START_REF] Tomlinson | Fgfr3 protein expression and its relationship to mutation status and prognostic variables in bladder cancer[END_REF]. However, the introduction of only K652E FGFR3 is not suicient to induce urothelial tumor in mice or to transform TERT-NHU cells [START_REF] Ahmad | K-ras and -catenin mutations cooperate with fgfr3 mutations in mice to promote tumorigenesis in the skin and lung, but not in the bladder[END_REF]. Only when this mutation iss combined with the deletion of PTEN, a negative regulator of PI3K-AKT, cell proliferation is increased [START_REF] Foth | Fibroblast growth factor receptor 3 activation plays a causative role in urothelial cancer pathogenesis in cooperation with pten loss in mice[END_REF].

Although the oncogenic property of mutated FGFR3 is well established, consequences of FGFR3 mutations in FGFR3 signaling in bladder cancer is partly understood. A recent work of our laboratory has revealed new elements in the signaling pathway activated by altered FGFR3 in bladder cancer [START_REF] Mahe | Caractérisation des voies de signalisation des oncogènes FGFR3 muté et FGFR3-TACC3 dans les carcinomes de vessie[END_REF]. Constitutively activated FGFR3 phosphorylates P38 that results in stabilization of MYC mRNA by translocating Human antigen R protein into the nucleus. On the other hand, the FGFR3-induced AKT phosphorylation inhibits Glycogen synthase kinase 3 (GSK3 ) which prevents proteasome-mediated degradation of MYC protein (Figure 2.14). Together, it showed that constitutive FGFR3 activation results in the activation of a MYC transcription program. The study was performed on two bladder cancer cell lines presenting constitutively activated FGFR3: MGH-U3 cells express a mutated activated FGFR3 (Y375C) and RT112 cells show a high ampliication and upregulation of both wild-type FGFR3 and a fusion form of FGFR3, FGFR3-TACC3. The signaling pathway described above is identical in the two cell lines suggesting that diferent activated forms of FGFR3 have the same efects on P38 and AKT pathway [START_REF] Mahe | Caractérisation des voies de signalisation des oncogènes FGFR3 muté et FGFR3-TACC3 dans les carcinomes de vessie[END_REF].

Recently, TGF -activated kinase 1 (TAK1) has been identiied as a novel partner protein of FGFR3 [START_REF] Salazar | Fibroblast growth factor receptor 3 interacts with and activates tgf -activated kinase 1 tyrosine phosphorylation and nfb signaling in multiple myeloma and bladder cancer[END_REF] and was phosphorylated by FGFR3 in vitro. In MGH-U3, activated FGFR3 modulated NFB transcriptional activity in a TAK1-dependent manner. [START_REF] Mahe | Caractérisation des voies de signalisation des oncogènes FGFR3 muté et FGFR3-TACC3 dans les carcinomes de vessie[END_REF].

Alterations of FGFR3 in bladder cancer 35 Figure 2.14 Signaling pathways activated by altered FGFR3 in bladder cancer

FGFR3-TACC3 was generally upregulated in bladder cancer cell lines or tumor samples. These cell lines were more sensitive to FGFR inhibition than cell lines with point mutations. All FGFR3-TACC3 fusion proteins are constitutively phosphorylated probably due to the auto-dimerization via the coiled-coil domain of TACC3 [START_REF] Wu | Identiication of targetable fgfr gene fusions in diverse cancers[END_REF]. In immortalized normal human urothelial cells TERT-NHU expressing FGFR3-TACC3, ERK1/2 were phosphorylated, but not PLC due to a deletion of the last exon of FGFR3 which is the binding site of PLC [START_REF] Williams | Oncogenic fgfr3 gene fusions in bladder cancer[END_REF][START_REF] Singh | Transforming fusions of fgfr and tacc genes in human glioblastoma[END_REF].

Rat-2 ibroblast cells stably expressing FGFR3-BAIAP2L1 had an anchorage independent growth in a soft agar assay and in a spheroid formation assay transforming activity, and had a high tumorigenic potential in mice. These cells were sensitive to the selective FGFR kinase activity inhibitor CH5183284/Debio 1347, indicating that FGFR3 kinase activity is crucial for tumorigenesis. The ligand-independent dimerization of fusion protein was created through the BAR domain of BAIAP2L1. The gene signature analysis revealed that this constitutively activated FGFR3 kinase domain led to the activation of MAPK pathway and the inhibition of tumor-suppressive pathways involving RB, p53 and CDKN2A [START_REF] Nakanishi | Mechanism of oncogenic signal activation by the novel fusion kinase fgfr3-baiap2l1[END_REF]. Also, the study of Wu and colleagues [START_REF] Wu | Identiication of targetable fgfr gene fusions in diverse cancers[END_REF] suggested that FGFR3-BAIAP2L1 fusion protein induced ERK1/2 and STAT1 activation.

FGF-and FGFR-targeted therapy

On the basis of the evidence for the role of FGF signaling in tumorigenesis, so far three approaches of FGF-or FGFR-targeting anticancer agents have been generated, including small-molecule tyrosine kinase inhibitors, monoclonal antibodies and FGFligand traps.

Small-molecule kinase inhibitors. Kinase inhibitors are the agents targeting the ATP-binding site of the tyrosine kinase domain of FGFRs [START_REF] Brooks | Molecular pathways: ibroblast growth factor signaling: a new therapeutic opportunity in cancer[END_REF][START_REF] Desai | Fgfr signaling as a target for lung cancer therapy[END_REF]. The irst-generation of kinase inhibitors which has a weak FGFR inhibitory activity is brivanib, lenvatibib, ponatinib and nintedanib. They have instead dominant anti-vascular endothelial factor receptor (VEGFR) and anti-platelet derived growth factor receptor (PDGFR) activity. In the past few years, the second-generation compounds have been developed which are potent kinase inhibitors and selective to FGFRs versus others tyrosine kinase receptors. In vitro studies indicated that AZD4547 (AstraZeneca) and BGJ398 (Novartis) are more potent inhibitors of FGFR1-3 whereas LY2874455 (Eli Lilly) and JNJ-4275649 (Johnson and Johnson) are pan-FGFR inhibitors. Currently, AZD4547 is being introduced in phase II and III in non-small cell lung cancer; BGJ398 is being tested in phase II trials in patients having diferent tumor types and presenting FGFR genetic alterations; whereas, LY2874455 and JNJ42756493 are being tested in phase I trials in advanced solid tumors. Due to the physiological role of FGFR signaling pathway in normal phosphate and vitamin D homeostasis, hyperphosphatemia has been a big problem as a mechanism-based toxicity of potent FGFR inhibitors. Thus, the challenge for speciic FGFR inhibitors in the clinic is to determine a therapeutic dose that will balance anti-tumor eicacy with side efects [START_REF] Brooks | Molecular pathways: ibroblast growth factor signaling: a new therapeutic opportunity in cancer[END_REF].

Monoclonal antibodies and FGF-ligand traps. Monoclonal antibodies and FGFligand traps are being developed with the hope to have a higher selectivity to a particular FGF ligand or FGF receptor. Thus, these approaches may potentially reduce the side efects of FGFR kinase inhibitors. The irst FGFR antibody to be tested in clinical phase I trial is the anti-FGFR3 antibody MFGR1877S (Roche) in t(4;14) translocated multiple myeloma patients. Recently, anti-FGFR3 monoclonal antibody B-701 has been evaluated in a phase II trial study for patients with locally advanced or metastatic urothelial cell Carcinoma (NCT02402542). The FGF-trap, FP-1039 (GlaxoSmithKline), is a soluble fusion protein which consists of the extracellular FGFR1-IIIc domain fused to the Fc portion of IgG1. This FGF-trap prevents the binding of FGF1, FGF2, and FGF4 to their corresponding receptors. FP-1039 is currently being introduced in a phase II in patients with endometrial cancer carrying FGFR2 mutations [START_REF] Brooks | Molecular pathways: ibroblast growth factor signaling: a new therapeutic opportunity in cancer[END_REF].

Lessons from others growth-factor-targeting anticancer agents indicated that cancer cells frequently develop drug resistances to targeted monotherapy. Using a drug cocktail which targets diferent altered genes in cancer cells should be a good approach to reduce these resistances.

Endocytosis and signaling

3

In the previous chapter, I mentioned that FGFRs as well as other RTKs transfer the extracellular stimuli to the cellular interior through activating several signaling cascades that inally result in physiological responses. In the classical model of signal transduction, transmembrane receptors can recruit many downstream substrates to the plasma membrane to activate signaling pathways and endocytosis serves as receptor clearance pathway to attenuate receptor signaling. However, growing evidence supports the idea that endocytosis has many efects on signal transduction and conversely, the receptor signaling can regulate the endocytic traicking. In this chapter, I irst report general features of endocytosis at diferent steps during membrane traicking. Then, I summarize the current understanding of the mechanistic and functional principles underlying this bidirectional relationship, with focus on those of RTKs.

Intracellular traicking of transmembrane receptors

3.1.1

Endocytic uptake pathways

There are many distinct mechanisms for endocytosis and generally, one molecule can be internalized through several endocytic routes [START_REF] Doherty | Mechanisms of endocytosis[END_REF][START_REF] Kirchhausen | Molecular structure, function, and dynamics of clathrin-mediated membrane traic[END_REF][START_REF] Johannes | Building endocytic pits without clathrin[END_REF]. Figure 3.1 summarizes the ten endocytic pathways identiied to date. They can be divided into 2 groups: Clathrin-dependent endocytosis (CDE) and Clathrin-independent endocytosis (CIE).

Here, I only focus on the CDE and the caveolae-/caveolin 1-dependent pathway, which represents a well-documented CIE.

Clathrin-dependent pathway. CDE is well-established in terms of morphology, structure and participating molecules. Five steps have been identiied during the Clathrincoated vesicle (CCV) cycle, illustrated in Figure 3.2 (reviewed in [START_REF] Mcmahon | Molecular mechanism and physiological functions of clathrin-mediated endocytosis[END_REF][START_REF] Kirchhausen | Molecular structure, function, and dynamics of clathrin-mediated membrane traic[END_REF]). This cycle initiates with the nucleation step where a low curvature membrane invagination, socalled a pit, is formed. This formation requires membrane-bending activity and some molecules such as FCH domain only (FCHO) proteins and EGFR pathway substrate 15 (EPS15). During the second step, AP2 and other cargo-speciic adaptor proteins are recruited to the pit to speciically promote cargo selection. AP2 can bind to PIP2

Other proteins implicated

Clathrin, AP2, epsin, SNX9, synaptojanin, actin amphiphysin, plus many others Caveolins, PTRF, src, PKC, actin (many signaling proteins localize to these sites)

a Abbreviations: CLIC, clathrin-independent carrier; GEEC, GPI-AP enriched early endosomal compartment; GPCRs, G protein-coupled receptors; GPI, glycosylphosphatidylinositol; MHC, major histocompatibility complex; RTK, receptor tyrosine kinase. and speciic motifs of transmembrane receptors through itsand -subunits; also, it interacts with the cargo via adaptor proteins. Some examples of cargo-speciic adaptor proteins can be cited, -arrestin recruits GPCRs to AP2 or ARH links LDLR to AP2. The next step is the clathrin coat assembly around the deined pit by AP2 and cargo-speciic adaptor proteins. A clathrin unit, the triskelion formed by three heavy chains associated with three light chains, is recruited from the cytosol to the deined pit at the plasma membrane. Clathrin units then assemble to form a curved lattice, which consequently stabilizes the curvature. Dynamin (DNM) is then recruited to the neck of the pit for CCV budding step. The polymerization of dynamin facilitates GTP hydrolysis and results in membrane ission. Following the detachment from the plasma membrane, the vesicle uncoats the clathrin lattice by the activity of ATPase heat shock cognate 70 (HSC70) and its cofactors (auxilin and GAK). The clathrin machinery, therefore, comes back to the cytosol for another CCV cycle [START_REF] Mcmahon | Molecular mechanism and physiological functions of clathrin-mediated endocytosis[END_REF]. CDE has been virtually identiied as the major route of internalization for every family of signaling receptors and this hypothesis has been mostly proven under physiological conditions (for example, RTKs, Notch receptors, integrins or G protein-coupled receptors (GPCRs) [START_REF] Sorkin | Endocytosis and signalling: intertwining molecular networks[END_REF]. Uncoating: auxilin or cyclin G-associated kinase (GAK) recruit HSC70 to disassemble the clathrin coat and produce an endocytic vesicle containing the cargo molecules. Synaptojanin probably facilitates this by releasing adaptor proteins from the vesicle membrane through its PtdIns lipid phosphatase activity. The components of the clathrin machinery are then freed and become available for another round of CCV formation [START_REF] Mcmahon | Molecular mechanism and physiological functions of clathrin-mediated endocytosis[END_REF].

Caveolae-/caveolin 1-dependent pathway. Caveolae are small invaginations, which have a morphology close to clathrin-coat pits and are enriched in cholesterol and sphingolipids (reviewed in [START_REF] Lamaze | Caveolae and cancer: A new mechanical perspective[END_REF][START_REF] Mayor | Clathrin-independent pathways of endocytosis[END_REF]). This endocytic pathway has been initially described to function in membrane traicking, cell signaling and recently, to serve as plasma membrane sensors to mechanical stimuli [START_REF] Sinha | Cells respond to mechanical stress by rapid disassembly of caveolae[END_REF]. Caveolin (CAV1, 2, 3) and cavins (Cavin 1, 2, 3, 4) are major players for the generation of caveolae structure. This pathway is dependent on dynamin which is localized to the neck of the caveolae and functions as budding actor. In contrast, EHD2, an ATPase, is required to stabilize the caveolae at the plasma membrane. In addition, EHD2 interacts with Pacsin2 which is able to sense and modulate membrane curvature. To date, it is known that albumin, SV40, cholera toxin B subunit, 1-integrin and glycosylphosphatidylinositol (GPI)-linked proteins use the caveolae-dependent endocytosis to enter the cell [START_REF] Doherty | Mechanisms of endocytosis[END_REF].

3.1.2

Post-endocytic traicking

Early endosome as a cargo-sorting station

Following internalization through either CDE or CIE, receptors reach the Early endosomes (EE) as the irst sorting station. RAB5, EEA1, PI3K and its product Phosphatidylinositol-3-phosphate (PI3P) are markers of EE. EE are large vesicles (∼400 nm) containing regions of thin tubular extensions (∼60 nm diameter). They are highly dynamic and able to fuse each other. The unique morphology of EE is thought to be important for the subsequent cargo sorting [START_REF] Jovic | The early endosome: a busy sorting station for proteins at the crossroads[END_REF]. From EE, receptors can be delivered to three destinations, illustrated in Figure 3.4 [START_REF] Scita | The endocytic matrix[END_REF]. The irst destination is the fast recycling route back to the plasma membrane in which RAB4 and RAB35 have been identiied as being im- The main molecules that shape caveolae (caveolins, cavins and pacsin2) and the proteins that regulate their dynamics (Dyn2, EHD2 and ilamin A) are depicted. The caveolar molecules that have functional or physical association with the actin cytoskeleton, and therefore potentially mediate the physical and functional interaction between caveolae and actin ibers, are also indicated. Filamin A is depicted here as the main protein mediating a linkage with stress ibers, but other yet unidentiied linkers might exist (indicated as unknown linker). Regulators of stress ibers (Abl kinases and mDia1) that impinge on caveolae organization and traicking are shown next to RhoA, the main regulator of stress ibers, which is regulated by Cav1 [START_REF] Echarri | Caveolae -mechanosensitive membrane invaginations linked to actin ilaments[END_REF].

portant for recycling of Transferrin receptor (TFRC) (t1/2 = 2 min). Second, transmembrane receptors are sorted to a later, mainly juxtanuclear compartment, well-known as endocytic recycling compartment, from which recycling endosomes are generated and come back to the plasma membrane. Third, ubiquitylated receptors can be transfered to multivesicular bodies before being delivered to late endosomes and lysosomes for degradation. Indeed, ubiquitylated receptors are recognized by multiprotein complexes, known as Endosomal sorting complex required for transport (ESCRT) (0, I, II and III), which facilitates the entry of receptors to multivesicular bodies [START_REF] Raiborg | The escrt machinery in endosomal sorting of ubiquitylated membrane proteins[END_REF]. Traficking between the endosomal compartments is spatiotemporally controlled by small RAB GTPases (further described in Chapter 4). An important feature of the endosomal sorting is the speciic targeting to each destination. The mechanism specifying the intracellular traicking of RTKs can be dependent on the ligand for a given receptor, RTK type for a given ligand, co-receptor for ligand-RTK complexes and speciic sequences on RTK [START_REF] Miaczynska | Efects of membrane traicking on signaling by receptor tyrosine kinases[END_REF].

Late endosomes and lysosomes

Late endosomes (LE) are composed of round or oval vesicles having a diameter of 250-1000 nm. LE are characterized by the presence of LAMP1 at its membrane and a component of acid hydrolases in the lumen (Figure 3.5). The lumen contains numer- ous intralumenal vesicles with a diameter of ∼ 50-100 nm and has a pH in the 6.0-4.8 range [START_REF] Huotari | Endosome maturation[END_REF]. The transition from early to LE is regulated by the crosstalk between RAB5, key player of EE and RAB7, key player of LE. It was shown that RAB7 recruits HOPS complex in which VPS39 subunit is a RAB7 GEF in a RAB5-dependent manner. Mathematical prediction suggests that RAB5 activates RAB7 until the latter reaches a threshold upon which it inactivates RAB5 via a negative-feedback loop. This loop recruits a RAB5 GAP in a RAB7-dependent manner [START_REF] Stenmark | Rab gtpases as coordinators of vesicle traic[END_REF]. Late endocytic compartments were initially considered as platforms for degradation, resulting in attenuate receptor signaling. However, MAPK scafold complex is detected at LE, suggesting that signaling continues even receptors are sorted to a degradation pathway [START_REF] Stenmark | The rabs: a family at the root of metazoan evolution[END_REF].

Endocytic recycling pathways

Due to technical challenges, the fast recycling route is still poorly understood. Here, I only discuss about the slow recycling routes that require the full function and positioning of the endocytic recycling compartment (ERC).

The ERC is composed of a tubular network with a diameter of about 60 nm where recycling endosomes are generated. The ERC network is closely associated with microtubule and actin cytoskeleton. In some cell types, the ERC is mainly condensed at the perinuclear region around the centrosome, but in other cells, this network is distributed more widely throughout the cytoplasm [START_REF] Maxfield | Endocytic recycling[END_REF]. Figure 3.6 illustrates the role of RAB11 and other proteins in the control of endocytic recycling. The RAB11 GT-Pase subfamily and their efectors play a crucial role in the traic to and from the ERC (further detail in the section 4.2). Along with these proteins, ARF6 and their efectors are thought to be important actors of recycling pathway of CIE cargoes through their Figure 3.5 The endosome/lysosome system. The primary endocytic vesicles deliver their contents and their membrane to EE in the peripheral cytoplasm. After a period of about 8-15 min during which the EE accumulate cargo and support recycling to the plasma membrane (directly or via recycling endosomes in the perinuclear region), conversion of the EE to LE takes place. Thus, as the endosomes are moving towards the perinuclear space along microtubules (MT), the nascent LE are formed inheriting the vacuolar domains of the EE network. They carry a selected subset of endocytosed cargo from the EE, which they combine en route with newly synthesized lysosomal hydrolases and membrane components from the secretory pathway. They undergo homotypic fusion reactions, grow in size, and acquire more intralumenal vesicles. Their role as feeder system is to deliver this mixture of endocytic and secretory components to lysosomes. To be able to do it, they continue to undergo a maturation process that prepares them for the encounter with lysosomes. The fusion of an endosome with a lysosome generates a transient hybrid organelle, the endolysosome, in which active degradation takes place. What follows is another maturation process; the endolysosome is converted to a classical dense lysosome, which constitutes a storage organelle for lysosomal hydrolases and membrane components [START_REF] Huotari | Endosome maturation[END_REF].

Bidirectional link between membrane traicking and signaling 43

implication in membrane fusion with the plasma membrane or interactions with the actin cytoskeleton, which relects the role of ARF6 in cell spreading, cell migration and metastasis. For example, the recycling of the syndecan 1 and FGFR complex required ARF6, PIP2 and syntenin which can bind to the C-terminus of syndecan. Introduction of diferent syntenin mutants, that can not bind to PIP2, in MCF-7 cells leads to the accumulation of syndecan and FGFR in the cells. Inhibiting syndecan recycling back to the plasma membrane results in impaired cell spreading [START_REF] Zimmermann | Syndecan recycling [corrected] is controlled by syntenin-pip2 interaction and arf6[END_REF]. This is one of the examples showing that the traicking of internalized receptor back to the plasma membrane can inluence its signaling. . Both types of cargo can move from the early endosome to the ERC by a process that requires sorting nexin 4 (SNX4), dynein, RAB10, RAB22A, RAB11FIP2 (in complex with carboxy-terminal epidermal growth factor receptor substrate 15 homology domain-containing protein 3 (EHD3)), RAB11FIP3 (in complex with ARF6 or RAB11) and RAB11FIP5. From the ERC, recycling of both types of cargo requires RAB11, and recycling of clathrin-independent cargoes involves the generation of distinctive RAB8-and RAB22Adependent tubules, in addition to many other factors. Some clathrin-dependent cargoes might also recycle through these diferent pathways. RAB10, RAB11, RAB22A and RAB35 are associated with the tubular recycling endosome and, along with EHD1 (homologous to receptor mediated endocytosis protein 1 (RME-1) in C. elegans) in complex with the mammalian apoptosis-linked gene 2-interacting protein X1 homologue ALIX (also known as PDCD6IP) and RAB11FIP2, are required for recycling. In the periphery, the tubules seem to break up into vesicles prior to fusing with the cell surface, a process that initially requires partitioning defective protein 3 (PAR3) and CDC42 and subsequently ADP-ribosylation factor 6 (ARF6), RAB11 and cortical actin. This is a composite description of endocytic recycling and all of the components shown here may not be evident in a given cell type. Adapted from [START_REF] Grant | Pathways and mechanisms of endocytic recycling[END_REF].

Bidirectional link between membrane traicking and signaling

Numerous indings with diferent types of receptors support the concept of a bidirectional link between membrane traicking and signaling. Here, I focus on those described for RTKs.

3.2.1

Receptor traicking regulates RTK signaling

In the classical model, following ligand stimulation, activated RTKs generate multiple docking sites for adaptor and efector proteins, thus enabling the activation of various signaling pathways, for example, the MAPK cascades or PI3K-AKT pathway. It has long been considered that ligand-bound endocytosis is one of the main ways for negatively regulating signaling from the cell surface. This negative regulation is executed by physically reducing the concentration of transmembrane receptor accessible to the ligand, and by separating the receptors from substrates and adaptors exclusively localized at or near the plasma membrane. However, it is now clear that signal transduction of various receptors can occur from diferent endosomal compartments (extensively reviewed in [START_REF] Sorkin | Endocytosis and signalling: intertwining molecular networks[END_REF][START_REF] Platta | Endocytosis and signaling[END_REF][START_REF] Miaczynska | Efects of membrane traicking on signaling by receptor tyrosine kinases[END_REF]). Indeed, endosomes provide an excellent platform for signal transduction because of their unique features including: a small volume facilitating ligand-receptor association; a residence of active receptors for a long duration; a fast capacity of motility over long distance by using microtubules; the presence of speciic resident proteins. Two types of signaling at endosomes, hereafter called endosomal signaling, have been identiied: irst, signaling initiates at the plasma membrane and continues in endosomes; second, signaling occurs only in endosomal compartments.

Continuous RTK signaling in endosomes

In systems in which active receptors are rapidly internalized into endosomes, endosomal signaling is important to ensure the duration of signaling. The irst evidence was reported in the work of Vieira and colleagues in which ERK1/2 activation requires the internalization of EGFR for full activation [START_REF] Vieira | Control of egf receptor signaling by clathrin-mediated endocytosis[END_REF]. Recent evidence also supports this concept [START_REF] Platta | Endocytosis and signaling[END_REF], such as the fact that several RTKs remain bound to their ligand, phosphorylated and active in endosomes until late stages. In addition, a wide range of components of MAPK cascades including GRB2, SHC, SOS, Ras proteins, RAF, MEK1 and MEK2, are detected in diferent endosomes. This collection of adaptor and efector proteins in endosomes ensures the activation of diferent cascades. The existence of a MAPK scafold complex in late endosomes is in favor of the requirement of endocytosis for the full activation of ERK1/2. For example, the phosphorylated p14 and MP1 recruit MEK1 to late endosomes to continue EGFR-initiated signaling for early embryogenesis and tissue homeostasis (Figure 3.7.a) [START_REF] Teis | p14-mp1-mek1 signaling regulates endosomal traic and cellular proliferation during tissue homeostasis[END_REF].

Endosomes with their capacity to move over a long distance are ideal for a precise, directional delivery of signaling into their site of action. For example, neuron Trk receptors (NTRK) are activated by NGF ligand which requires endocytosis and retrograde transport for the full activation of ERK5 and phosphorylation of the cAMP responsive element-binding protein (CREB) in the neuron soma [START_REF] Watson | Neurotrophins use the erk5 pathway to mediate a retrograde survival response[END_REF]. To ensure an eicient signaling, endosomes containing NGF-bound Trk are delivered from distal axonal termini to the cell body in a microtubule-mediated manner [START_REF] Wu | A functional dynein-microtubule network is required for ngf signaling through the rap1/mapk pathway[END_REF]. In addition, these endosomes contain PLC , PI3K, MEK1, p38, B-Raf, Gab2 and Rap1 which are involved in the activation of ERK signaling (Figure 3.7.b) [START_REF] Miaczynska | Efects of membrane traicking on signaling by receptor tyrosine kinases[END_REF]. In all of these locations activated, phosphorylated MAPK dissociates from the MAPK kinase (MK), unless stably associated with a scafold protein such as betaarrestin, and phosphorylates substrates in the cytoplasm and nucleus. When GPCRs are stimulated by lysophosphatidic acid, the activated G protein subcomplex Gbeta-Ggamma can activate PI3K and Akt at the plasma membrane and also translocate to the early and recycling endosomes to activate endosomal PI3K gamma-isoform, which leads to the phosphorylation and activation of Akt in endosomes. b Model of retrograde endosome signaling in neurons. Ligand-bound nerve growth factor receptors (NTRKs) are internalized by clathrin-dependent and clathrin-independent endocytosis, with associated components of the MAPK signaling cascade, into early endosomes or macropinosomes that have a multivesicular body-like morphology. NTRK signaling complexes are delivered to the soma in retrograde endosomal carriers, which are a population of early or late endosomes and macropinosomes, by dynein motor-mediated microtubular transport. The MAPK extracellular signal-regulated kinase 5 (ERK5) is then phosphorylated and activated in the soma. It phosphorylates the cAMP responsive element-binding protein (CREB), which regulates the transcription of anti-apoptotic genes. Grey arrows show traicking pathways. Black arrows show protein translocations or complex assembly. GRB2, growth factor receptor-bound protein 2; MP1, MEK1 partner 1; PtdIns(3,4,5)P3, phosphatidylinositol-3,4,5-trisphosphate; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; SH2, Src homology 2. [START_REF] Sorkin | Endocytosis and signalling: intertwining molecular networks[END_REF].

Speciic signaling in endosomes

In some cases, endosomes serve as a unique platform for the recruitment of speciic signaling substrates to amplify signals of activated RTK. An example is the activation mechanism of STAT3 by HGF [START_REF] Kermorgant | Receptor traicking controls weak signal delivery: a strategy used by c-met for stat3 nuclear accumulation[END_REF]. STAT3 is weakly activated by HGF stimulation on c-Met receptor; however, increased phosphorylation and subsequent nuclear translocation of STAT3 was observed when activated Met was delivered to the perinuclear endosomes.

Another example is the case of APPLs-mediated AKT recruitment on EE. APPLs (APPL1, APPL2) are RAB5 efectors, and can interact with EGFR or other RTKs on EE [START_REF] Miaczynska | Appl proteins link rab5 to nuclear signal transduction via an endosomal compartment[END_REF]. APPLs can recruit AKT which is mostly localized to the plasma membrane. This results in the activation of GSK3 but not TSC2 for prosurvival response. Indeed, a small pool AKT and GSK3 is found to be transiently associated with RAB5 and APPLpositive endosomes [START_REF] Schenck | The endosomal protein appl1 mediates akt substrate speciicity and cell survival in vertebrate development[END_REF]. Together, APPLs-positive endosomes provide a platform for selective recruitment and activation of signaling components.

Balance between degradation and recycling of RTKs inluences their signaling

The balance between degradation and recycling of a given RTK has an important impact on the signal quality, duration, and magnitude [START_REF] Lemmon | Cell signaling by receptor tyrosine kinases[END_REF]. Generally, ubiquitinylated EGFR is delivered to late endosomes then to lysosomes. Inhibiting EGFR ubiquitinylation by mutating ubiquitin sites on the receptor induces EGFR signaling, and conversely increasing ubiquitinylation downregulates signaling [START_REF] Sorkin | Endocytosis and signalling: intertwining molecular networks[END_REF]. Moreover, ESCRT complexes are essential for the maturation of late endosomes to lysosomes. It was shown that the depletion of one of these complexes is enough to impair EGFR degradation, leading to enhancing EGFR-induced ERK signaling [START_REF] Platta | Endocytosis and signaling[END_REF]. Another example is FGF signaling negatively controlled by SRED2. SPRED2 was shown to interact and colocalize with NBR1, a late endosome resident protein. This association leads to diverting FGFR into the lysosomal degradation pathway. Also, the inhibition of ERK1/2 signaling by SPRED2 is dependent on its interaction with NBR1 [START_REF] Mardakheh | Spred2 interaction with the late endosomal protein nbr1 down-regulates ibroblast growth factor receptor signaling[END_REF]. The endocytic recycling plays an important role in sustaining receptor signaling. For example, in cells expressing FGFR4, blocking the recycling pathway by depletion of RAB11 induces the prolongation of phosphorylation of PLC but reduced the phosphorylation of AKT and ERK [START_REF] Haugsten | Photoactivation approaches reveal a role for rab11 in fgfr4 recycling and signalling[END_REF]. The molecular mechanism however remains unknown. Also, it was shown that Met receptor can sustain ERK1/2 activation from endosomes and the receptor recycling plays a major role in the full activation of ERK1/2 [START_REF] Joffre | A direct role for met endocytosis in tumorigenesis[END_REF].

3.2.2

RTK signaling regulates endocytosis

Although this regulation remains poorly understood, several recent evidence supports this idea. Early evidence came from the fact that ligand-induced activation of EGFR increases the total number and surface density of clathrin-coated pits. This event was then shown to be dependent on the Src kinase activity [START_REF] Sorkin | Endocytosis and signalling: intertwining molecular networks[END_REF]. Similarly, Src plays a role in clathrin-mediated FGFR2 internalization. In addition, Eps8, phosphorylated by Src, is required for FGFR internalization and intracellular traicking. Inhibiting Src activity by Src kinase inhibitor or depletion of Eps8 reduces clathrin-mediated FGFR2

Bidirectional link between membrane traicking and signaling 47

internalization. Eps8 is important for the traicking of FGFR into early endosome and out of this compartment [START_REF] Auciello | Regulation of ibroblast growth factor receptor signalling and traicking by src and eps8[END_REF].

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RAB GTPase family

4

RAB proteins constitute the largest family of small GTPases with 11 Rab identiied in yeast (also called Ypt) and 70 members in human. RAB GTPases virtually regulate all transport steps between cell compartment. Therefore, alterations in RAB functions are directly or indirectly responsible for a wide range of diseases. In particular, growing evidence points out their roles in cancer development and progression [START_REF] Stenmark | Rab gtpases as coordinators of vesicle traic[END_REF][START_REF] Kelly | Rab11 proteins in health and disease[END_REF].

4.1

Role of RAB in intracellular transport

4.1.1

Structural characteristics

RAB proteins are typically 20-25 kDa monomers and share a common core fold with other small GTPases of the Ras superfamily (Figure 4.1). The fold contains a sixstranded -sheet lanked by ive -helices. RABs distinguish however from other members of the Ras superfamily through ive conserved RAB speciic sequences, RABF1-F5 and four conserved sub-family motifs, RABSF1-4 [START_REF] Pfeffer | Structural clues to rab gtpase functional diversity[END_REF].

In terms of functional features, RAB contains two "switch regions" called Switch I and Switch II that are responsible for guanine nucleotide and Mg2+ binding, as well as GTP hydrolysis. Following GTP binding, the switch regions undergo conformational changes that allow binding of RAB efectors. The hypervariable region at the C-terminus of RAB was originally proposed to be involved in targeting RABs to speciic membranes. However, growing evidence indicates that RAB targeting relies on other mechanisms, in particular on interactions with Guanine nucleotide Exchange Factor (GEF) proteins [START_REF] Kelly | Rab11 proteins in health and disease[END_REF]. The extreme C-termini contain a cysteine-motif box that can exist as several forms (CXXX, CXC, CCXX or CCXXX; where X is any other amino acid). One or two cysteine residues are geranylgeranylated, which allows RABs to stably insert in the outer lealet of intracellular membranes [START_REF] Hutagalung | Role of rab gtpases in membrane traic and cell physiology[END_REF]. A proile amino-acid sequence of the RAB GTPase subfamily generated using the hidden Markov model (HMM) method. The uppercase/lowercase coding represents outcome of the proile HMM method. Upper-case characters, residues found in the proile with a probability of p<0.5; red, RAB-speciic residues (RABF1-5); dark blue, subfamily-speciic motifs (RABSF1-4); cyan, highly conserved nucleotide-binding motifs; G, guanine-base-binding motif; PM, phosphate/magnesiumbinding motif. The secondary structure units ( helices, strands, and loops, ) are indicated above the sequence [START_REF] Stenmark | The rab gtpase family[END_REF].

Role of RAB in intracellular transport 51

4.1.2

The RAB cycle

Like other small GTPases, RABs act as molecular switches by oscillating between an inactive GDP-bound form and an active GTP-bound form (Figure 4.2) [START_REF] Hutagalung | Role of rab gtpases in membrane traic and cell physiology[END_REF][START_REF] Kelly | The rab family of proteins: 25 years on[END_REF]. The nucleotide-bound states of RABs determine its localization and activity. A newly synthesized RAB associates with RAB escort protein (REP) and is geranylgeranylated by RAB geranylgeranyl transferase (RABGGT). REP then delivers the geranylgeranylated, GDP-bound RAB to target membranes where GTP/GDP exchange takes place thanks to guanine nucleotide exchange factor (GEF). The active GTP-bound RAB then recruits efector proteins including sorting adaptors, tethering factors, kinases, phosphatases and molecular motors to fulill their speciic functions in membrane traicking.

To terminate the cycle, RABs interacts with GTPases activating protein (GAP) which initiates GTP hydrolysis to obtain the inactive GDP-bound state. Guanine exchange dissociation inhibitor (GDI)) then allows extraction of RABs from membranes. The RAB:GDI is now ready to be reinserted into a membrane and begin a new cycle.

The RAB cycle is critical for ensuring speciicity and vectoriality of intracellular transport. that then directs it to RAB geranylgeranyl transferase (RabGGT) to receive its prenyl tails (red wavy lines). REP delivers the RAB to its target membrane. Throughout this process, the RAB is GDP-bound. A guanine nucleotide exchange factor (GEF) catalyzes exchange of GDP for GTP to activate the RAB. The GTP-bound RAB interacts with efector proteins that mediate membrane traic in the pathway regulated by its associated RAB. The RAB then interacts with its associated GTPase activating protein (GAP) that catalyzes hydrolysis of GTP to GDP by the RAB. The RAB is then removed from the membrane by guanine nucleotide dissociation inhibitor (GDI) in preparation for the next cycle. Loss-of-function mutations at each of the above steps produce disease phenotypes as indicated by the yellow text boxes [START_REF] Hutagalung | Role of rab gtpases in membrane traic and cell physiology[END_REF].

4.1.3

Downstream efectors and related functions

RAB GTPases are key determinants in all steps of membrane transport because of their capacity in recruiting a wide range of diverse efectors. Figure 4.3 illustrates the main classes of RAB efectors and their role in RAB-mediated processes. To date, more than 300 RAB efectors have been identiied and the list still keeps getting longer [START_REF] Hutagalung | Role of rab gtpases in membrane traic and cell physiology[END_REF]. Below, I provide some examples of several well-studied RAB:RAB efector couples in regulating cargo selection, uncoating, motility, tethering and fusion.

Cargo selection. Transport from one compartment to the other is thought to be regulated by vesicular cargoes which are selectively formed by the assembly of coat protein complexes at donor membrane. For example, clathrin, AP2 and dynamin are involved in clathrin-dependent endocytosis; coatomer protein type I (COPI) is involved in membrane traicking between ER and the Golgi apparatus. Along with coat proteins in cargo selection, RAB proteins also contribute to ensuring the recruitment of speciic coat machinery to distinct membrane. The role of RAB9 in the transport of mannose-6-phosphate receptors (M6PRs) is a good example [START_REF] Li | Rab GTPases and membrane traicking[END_REF]. M6PRs function as d. RAB GTPases can mediate vesicle tethering by recruiting rod-shaped tethering factors that interact with molecules in the acceptor membrane. Such factors might interact with SNAREs and their regulators to activate SNARE complex formation, which results in membrane fusion. e. Following membrane fusion and exocytosis, the RAB GTPase is converted to its inactive GDP-bound form and comes back to the donor membrane [START_REF] Stenmark | Rab gtpases as coordinators of vesicle traic[END_REF].

Role of RAB in intracellular transport 53

carriers to deliver newly synthesized lysosomal enzymes from TGN to the lysosome. Thus, M6PRs are mainly transported from Golgi to early endosomes then to either the cell surface or late endosomes, then it is required to be recycled back to TGN for a new cycle. RAB9 which generally resides on late endosomes, functions in this retrograde transport of M6PRs. RAB9:GDI is irst recruited from the cytosol onto late endosome membranes where it is activated by a GEF. RAB9 efector, Tail interacting protein of 47kDa (TIF47), is then recruited onto the membrane by activated RAB9. RAB9:TIP47 complexes then bind to the C-terminal of M6PRs that creates a speciic and stable microdomain. Therefore, RAB9 enables the formation of nascent M6PR vesicles ready to bud from late endosome [START_REF] Li | Rab GTPases and membrane traicking[END_REF].

Vesicle uncoating. Intracellular traicking mainly relies on the formation of coated vesicle from donor membranes. These coats must be shed before the vesicles reach their target membranes. RAB5 has been shown not only to be necessary for the formation of clathrin-coated vesicles but also for the subsequent uncoating process. Once the clathrin-coated vesicles are formed from donor membrane, RAB5 is activated by hRME-6 and binds to AP2. This association of RAB5 and AP2 displaces 2 kinase from AP2. The 2 subunit of AP2 complex is consequently dephosphorylated and the level of PIP2 on the vesicles decreases. In addition, RAB5 can also recruit phosphoinositide 3 kinases or phosphoinositol phosphatases for the turnover of PIP2 [START_REF] Shin | An enzymatic cascade of rab5 efectors regulates phosphoinositide turnover in the endocytic pathway[END_REF].

Vesicle motility. Actin cytoskeleton and microtubules are essential actors in intracellular vesicle motility. Actin cytoskeleton serves as tracks for vesicle motility via molecular motors of the myosin family. The transport vesicles can also move along microtubules toward the cell interior or the cell periphery via the interaction with the dynein-dynactin complex or kinesins, respectively. Many studies have revealed the capacity of RAB proteins to recruit molecular motors on transport vesicles. Of note, a given RAB can interact with several motors, as exempliied in the case of RAB11A.

RAB11A is a member of the RAB11 family that includes three proteins (RAB11A, RAB11B, RAB25) encoded by three distinct genes. These proteins are implicated in the recycling process of many cell surface receptors and in cytokinesis [START_REF] Kelly | Rab11 proteins in health and disease[END_REF]. RAB11A recruits RAB11 family-interacting protein 2 (RAB11FIP2) which serves as an adaptor for Myosin Vb (MYO5B) [START_REF] Lapierre | Myosin vb is associated with plasma membrane recycling systems[END_REF]. Another studies indicates that MYO5B can directly bind to RAB11A in the endocytic recycling pathway [START_REF] Roland | Myosin vb interacts with rab8a on a tubular network containing ehd1 and ehd3[END_REF]. Recently, Delavoye et al [START_REF] Delevoye | 4 Bibliography[END_REF] reported that active GTP-bound form of RAB11A binds to microtubule motor KIF13A to regulate peripheral transport of recycling endosome tubules. I come back to this family in further detail in the section 4.2.

Vesicle tethering and fusion.

To ensure the idelity of membrane transport, the recruitment of tethering and fusion factors is essential. Fusion is mediated by the proteins of the SNARE family and RABs are involved in the recruitment of many tethering complexes that dock transport vesicles to their target membranes. For example, the RAB5 machinery has been shown to function in eicient fusion from endocytic vesicles to early endosomes as well as in clustering early endosomes together [START_REF] Christoforidis | Phosphatidylinositol-3-oh kinases are rab5 efectors[END_REF][START_REF] Rubino | Selective membrane recruitment of eea1 suggests a role in directional transport of clathrin-coated vesicles to early endosomes[END_REF][START_REF] Ohya | Reconstitution of rab-and snare-dependent membrane fusion by synthetic endosomes[END_REF]. Rabaptin-5-Rabex-5, Early endosome antigen 1 (EEA1), Rabenosyn-5 and hVPS34-PI3KR4 are essential efectors for the functional RAB5 machinery. Rabex-5, a RAB5 GEF, forms a tight physical complex with Rabaptin-5, a RAB5 efector to activate and stabilize the active state of RAB5. Active RAB5 (GTP-bound) in turn recruits Early endosome antigen 1 (EEA1) and Rabenosyn-5. These RAB5 efectors contain at the C-terminus a RAB 5 binding domain (R5BD); and a FYVE domain, a conserved phosphotidylinositol 3-phosphate (PI3P) binding motif. Interestingly, EEA1 plays a role in membrane docking thanks to another R5BD at its N-terminus that allows its interaction with another RAB5-GTP on fusion membranes. EEA1 and Rabenosyn-5 are important for the coordination of tethering and fusion. On one hand, Rabenosyn-5 directly interacts with vacuolar protein sorting-associated protein 45 (VPS45), a SNARE regulator. On the other hand, EEA1 and Rabenosyn-5 directly interact with early endosomal SNAREs including Syntaxin 13 and Syntaxin 6. Moreover, RAB5 recruits a PI-3-OH kinase VPS34 and PI(4)-and ( 5)-phosphatases to ensure the production of PI(3)P [START_REF] Christoforidis | Phosphatidylinositol-3-oh kinases are rab5 efectors[END_REF].

4.1.4

RAB GTPases and diseases

Since RAB GTPases play an important role in membrane traicking, these proteins and efectors have been reported to be associated with many diseases including infectious diseases, inherited disorders and cancers. For example, Helicobacter pylori secretes a toxin that sequesters RAB7 and creates a protective niche for intracellular survival of the bacterium. Mutations in RAB7 are associated with neuropathies such as Charcot-Marie-Tooth disease type 2B. RAB25 is found dysregulated in many cancers such as breast cancer and ovarian cancer. An extensive review about the roles of GTPases in diseases can be found in [START_REF] Stenmark | Rab gtpases as coordinators of vesicle traic[END_REF] and particularly those in cancer [START_REF] Chua | The role of the small gtpase rab31 in cancer[END_REF]. A systematic analysis in bladder cancer was performed in our laboratories to investigate the deregulation of expression of 61 RAB genes and 223 genes encoding RAB-interacting proteins [START_REF] Ho | Deregulation of rab and rab efector genes in bladder cancer[END_REF].

The RAB11 subfamily

Below, I review our current understanding of the RAB11 family, which is involved in membrane traicking of a wide range of receptors and identiied as a key player in many human disorders.

The three members of the RAB11 (RAB11A, RAB11B and RAB25) share a high sequence homology (RAB11A:RAB11B, 91% identity; RAB11A:RAB25, 62% identity; RAB11B:RAB25, 61% identity). Of note, RAB25 contains WDTAGLE motif, instead of WDTAGQE, which results in a dominant active phenotype [START_REF] Goldenring | Identiication of a small gtp-binding protein, rab25, expressed in the gastrointestinal mucosa, kidney, and lung[END_REF]. RAB11 proteins serve as regulators in recycling traicking of many cell surface receptors and for endosomal membrane transport during cytokinesis [START_REF] Kelly | Rab11 proteins in health and disease[END_REF][START_REF] Schiel | Membrane dynamics during cytokinesis[END_REF].

4.2.1

Localization and functions

RAB11A is ubiquitously expressed and predominantly localized to the pericentriolar ERC. RAB11A was identiied as the irst small GTPase implicated in slow endo-cytic recycling pathway of transferrin through ERC [START_REF] Ullrich | Rab11 regulates recycling through the pericentriolar recycling endosome[END_REF]. From this discovery, many studies have revealed the role of RAB11A in the recycling pathway of a wide range of receptors, for example, 5 1 integrin, Langerin and FGFR4 [START_REF] Wang | Myosin vb mobilizes recycling endosomes and ampa receptors for postsynaptic plasticity[END_REF]. Depletion of RAB11A results in the accumulation of receptors in pericentriolar transferrin-positive ERC [START_REF] Powelka | Stimulation-dependent recycling of integrin beta1 regulated by arf6 and rab11[END_REF][START_REF] Gidon | A rab11a/myosin vb/rab11-ip2 complex frames two late recycling steps of langerin from the erc to the plasma membrane[END_REF][START_REF] Haugsten | Photoactivation approaches reveal a role for rab11 in fgfr4 recycling and signalling[END_REF]. RAB11A also colocalizes with TGN38, a trans-Golgi network (TGN) resident protein, and regulates transport from endosomes back to the TGN [START_REF] Wilcke | Rab11 regulates the compartmentalization of early endosomes required for eicient transport from early endosomes to the trans-golgi network[END_REF]. RAB11A is also involved in other physiological process such as cytokinesis [START_REF] Prekeris | A rab11/rip11 protein complex regulates apical membrane traicking via recycling endosomes[END_REF] and long-term potentiation [START_REF] Wang | Myosin vb mobilizes recycling endosomes and ampa receptors for postsynaptic plasticity[END_REF].

RAB11B was subsequently identiied [START_REF] Lai | Molecular analysis of mouse rab11b: a new type of mammalian ypt/rab protein[END_REF] and was also shown to localize to the ERC and essential for the eicient recycling of transferrin [START_REF] Schlierf | Rab11b is essential for recycling of transferrin to the plasma membrane[END_REF]. However, a number of reports have indicated that RAB11B plays a distinct role compared to RAB11A in the endocytic recycling pathway. In rabbit gastric parietal cells or MDCK cells, RAB11B localizes to cellular compartments distinct from RAB11A [START_REF] Lapierre | Rab11b resides in a vesicular compartment distinct from rab11a in parietal cells and other epithelial cells[END_REF]. RAB11A and RAB11B were suggested to function at overlapping but also in diferent steps of the recycling pathway of FGFR4 [START_REF] Haugsten | Photoactivation approaches reveal a role for rab11 in fgfr4 recycling and signalling[END_REF]. Recently, RAB11B but not RAB11A has been shown to be a key regulator of recycling of protease-activated receptor-1 (PAR1) [START_REF] Grimsey | Recycling and endosomal sorting of protease-activated receptor-1 is distinctly regulated by rab11a and rab11b proteins[END_REF]. The authors proposed a model for the diferential regulation of PAR constitutive recycling and basal degradation by RAB11B and RAB11A (Figure 4.4).

RAB25 was initially found to be expressed in epithelial tissue [START_REF] Casanova | Association of rab25 and rab11a with the apical recycling system of polarized madin-darby canine kidney cells[END_REF] and to colocalize with RAB11A in an apical pericentriolar endosomal compartment. The association of RAB25 and RAB11A with the apical recycling system is required to regulate transcytotic pathways in polarized epithelial MDCK cells [START_REF] Casanova | Association of rab25 and rab11a with the apical recycling system of polarized madin-darby canine kidney cells[END_REF]. Interestingly, in cultured A2780 cells highly expressing CLIC3, a late endosomal/lysosomal protein, RAB25 was found to localize not only to recycling endosomes but also to a CLIC3-positive late endosomal population [START_REF] Dozynkiewicz | Rab25 and clic3 collaborate to promote integrin recycling from late endosomes/lysosomes and drive cancer progression[END_REF]. The localization of RAB25 in these cells is distinct from RAB11/transferrin endosomes. Indeed, RAB25 helps active integrins to escape the degradation pathway and transport back to the plasma membrane. This process is shown to be required for efective cell invasion. Accordingly, RAB25 and CLIC3 were both shown to be associated with aggressive cancers.

RAB11 motor protein complexes

To execute its functions in recycling traicking or cytokinesis, RAB11 recruits distinct motor adaptors. RAB11 forms complexes with myosin for actin-ilament-dependent transport or kinesins and dynein for bidirectional movement along microtubule tracks. Here, I introduce some studies investigating the role of RAB11 complexes in endosomal recycling of receptors. The role of RAB11 in cytokinesis can be found elsewhere [START_REF] Schiel | Membrane dynamics during cytokinesis[END_REF][START_REF] Prekeris | Breaking up is hard to do -membrane traic in cytokinesis[END_REF].

To unravel the molecular mechanisms underlying RAB11-dependent membrane transport, many approaches have been developed. By yeast two-hybrid screening, three members of the RAB11 subfamily were identiied to interact with the C-terminus of myosin Vb (MYO5B) tail [START_REF] Lapierre | Myosin vb is associated with plasma membrane recycling systems[END_REF]. Proteomic screenings then allowed identifying an evolutionarily conserved family of RAB11-efectors which have been termed the RAB11 family-interacting protein (RAB11FIP) [START_REF] Kelly | Rab11 proteins in health and disease[END_REF]. The RAB11FIPs (or FIPs) share a common C-terminal domain that interacts with RAB11, the so-called the RAB11-binding In the absence of RAB11A expression (right panel), PAR1 constitutively internalizes, and a marked increase in receptor accumulation in early endosomes is observed, as basal degradation of PAR1 is inhibited. Interestingly, in the absence of RAB11B expression (middle panel), PAR1 exhibits enhanced basal degradation that is blocked by RAB11A and ATG5 depletion, suggesting that the receptor traics through an autophagic pathway before degradation in the autolysosome. These indings suggest that RAB11B and RAB11A serve distinct functions and regulate PAR1 recycling or basal degradation, respectively [START_REF] Grimsey | Recycling and endosomal sorting of protease-activated receptor-1 is distinctly regulated by rab11a and rab11b proteins[END_REF].

domain.

The best-studied RAB11-FIPs-motor protein complex is the RAB11-FIP2-MYO5B which has been implicated in a wide range of endosomal recycling processes. RAB11 interacts directly with the MYO5B globular tail domain, while the C-terminal MYO5B sequences interact with FIP2. MYO5B and FIP2 recognize the same binding area of GTP-bound RAB11A [START_REF] Welz | Orchestration of cell surface proteins by rab11[END_REF]. An interaction model has been proposed in which FIP2 dimerization and MYO5B dimerization require four available RAB11-GTP to form the tripartite RAB11-FIP2-MYO5B complex (Figure 4.5). Temporal and spatial characterization of the dynamic processes of the recycling pathway mediated by the RAB11-FIP2-MYO5B complex has been addressed in the work of Gidon and colleagues [START_REF] Gidon | A rab11a/myosin vb/rab11-ip2 complex frames two late recycling steps of langerin from the erc to the plasma membrane[END_REF]. The authors performed a combination of high resolution imaging techniques to investigate the recycling pathways of langerin, a C-type lectin receptor which is constitutively endocytosed and recycled. A working model has been proposed in which the formation of RAB11A and FIP2 is required to sort langerin toward the late steps of the recycling pathway. MYO5B then interacts with RAB11A which provides part of the force required to generate transport recycling vesicles (Figure 4.6). RAB11-GTP (green sphere) forms a complex with the microtubule minus-end directed dynein motor protein complex. The interaction is mediated by the interaction of the FIP3 adaptor protein with the DLIC1. The RAB11/protrudin/KIF5 complex represents a RAB11 microtubule plus-end directed motor protein complex. Of note is the adaptor protein protrudin, which belongs to the FYVE zinc inger family of membrane binding proteins; it interacts with RAB11 in its GDP bound form (RAB11-GDP, red sphere). The RAB11-GTP/FIP2/MyoVb complex allows RAB11 vesicles to slide along actin ilaments. The unconventional MyoVb actin motor protein interacts directly with both the FIP2 adaptor and the RAB11 protein [START_REF] Welz | Orchestration of cell surface proteins by rab11[END_REF].

In addition to MYO5B, the microtubule-based motor kinesin KIF13A has been recently shown to interact with active GTP-bound forms of RAB11 proteins [START_REF] Delevoye | 4 Bibliography[END_REF] in yeast two-hybrid and Förster resonance energy transfer (FRET) and luorescence lifetime imaging microscopy (FLIM) experiments. The overexpression of KIF13A induced a dramatic tubulation of RAB11A-and FIP1-positive compartments. Conversely, depletion of KIF13A leads to the accumulation of large transferrin-positive structures and reduces the number of transferrin containing tubules, that results in the decrease of recycled transferrin. Altogether, these results indicate that KIF13A is required to generate recycling endosomal tubules along microtubule tracks from the vacuolar sorting endosomes. This process is also dependent on RAB11 since in RAB11-depleted cells, KIF13A-positive recycling compartment tubules were signiicantly decreased compared to control [START_REF] Delevoye | 4 Bibliography[END_REF].

4.2.3

RAB11 subfamily in cancer

Given the physiological importance of RAB11 proteins and their efectors in membrane traicking of many receptors, it is found that this subfamily is associated with many cancers. For example, RAB11FIP1 acts as a negative regulator in ErbB2-mediated mammary tumor progression [START_REF] Boulay | Rab11-ip1c is a critical negative regulator in erbb2-mediated mammary tumor progression[END_REF]. RAB11FIP3 regulates breast cancer cell motility by modulating the actin cytoskeleton [START_REF] Jing | Rab11-ip3 is a rab11binding protein that regulates breast cancer cell motility by modulating the actin cytoskeleton[END_REF]. In particular, there is growing attention toward RAB25 owing to its appearance in a number of cancer screens in diferent studies (Table 4.1). Here, I outline the current knowledge regarding to the implication of RAB25 in several hallmarks of cancer and its mechanism of action underlying this relationship. Working model for chronological events implicated in the recycling of langerin, from recycling endosomes to the plasma membrane. TIRF temporal characterizations of molecular events that lead to the docking/fusion at the plasma membrane of the recycling vesicles bearing langerin, are summarized in the graphs. Curves represent the average of normalized luorescence levels in a speciic region of the plasma membrane where a double luorescent vesicle is detected, for each time point after temporal registration based on the langerin maximum luorescence level for normalization (Lang-RAB11A n = 15, Lang-RAB11-FIP2 n = 12 and Lang-RAB11-FIP2RBD n = 13) or on RAB11A maximum luorescence level for RAB11A-Myosin Vb and RAB11A-RAB11-FIP2 conditions (n = 10 and 12, respectively). Around 40 frames before and 40 frames after the temporal occurrence of these maximums, are then considered for both luorescence channels in order to obtain values close to baseline of luorescence intensities and are plotted here [START_REF] Gidon | A rab11a/myosin vb/rab11-ip2 complex frames two late recycling steps of langerin from the erc to the plasma membrane[END_REF].

The RAB11 subfamily 59

Expression of RAB25 in human cancers. RAB25 was shown for the irst time to be upregulated in breast and ovarian cancers in the work of Mills and colleagues [START_REF] Cheng | The rab25 small gtpase determines aggressiveness of ovarian and breast cancers[END_REF]. Since then, dysregulated expression of RAB25 has been reported in a range of human cancers, summarized in the Table 4.1. Strikingly, elevated expression of this GTPase was reported in estrogen receptor-and HE2-positive breast cancers but an apparent loss of expression was observed in ER-negative and progesterone receptor-negative tumors [START_REF] Cheng | The rab25 small gtpase determines aggressiveness of ovarian and breast cancers[END_REF][START_REF] Cheng | Loss of rab25 expression in breast cancer[END_REF][START_REF] Agarwal | The emerging role of the rab25 small gtpase in cancer[END_REF]. This suggests that RAB25 may play diferent roles in cancers, depending on the subtype. RAB25 in cell proliferation and apoptosis. The overexpression of RAB25 in ovarian cancer cells results in cell transformation in vitro and increases aggressiveness of cancer cells in vivo [START_REF] Cheng | The rab25 small gtpase determines aggressiveness of ovarian and breast cancers[END_REF]. Conversely, downregulation of RAB25 by siRNA or short hairpin RNA (shRNA) inhibits cell proliferation, increases apoptosis in vitro and decreases tumor growth in vivo xenograft models [START_REF] Cheng | The rab25 small gtpase determines aggressiveness of ovarian and breast cancers[END_REF][START_REF] Fan | Knockdown of rab25 expression by rnai inhibits growth of human epithelial ovarian cancer cells in vitro and in vivo[END_REF]. In contrast, in basal-and triple-negative breast tumor representing cells, expression of RAB25 decreased cell transformation properties including proliferation and anchorage-independent growth, and reduces tumor growth of xenografted breast carcinoma cells in nude mice [START_REF] Cheng | Loss of rab25 expression in breast cancer[END_REF][START_REF] Cheng | Tumor suppressor function of rab25 in triple-negative breast cancer[END_REF]. The mechanism of action of RAB25 in cell proliferation and apoptosis remains however poorly understood.

RAB25 in cell migration and invasion. RAB25 was identiied as a component of the invasive and metastatic signature of breast cancer cells in vivo and in vitro [START_REF] Wang | Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular proiling[END_REF]. The mechanisms by which RAB25 promotes invasive migration and tumor aggressiveness were extensively reported in the studies of the Norman group [START_REF] Caswell | Rab25 associates with alpha5beta1 integrin to promote invasive migration in 3d microenvironments[END_REF][START_REF] Caswell | Rab-coupling protein coordinates recycling of alpha5beta1 integrin and egfr1 to promote cell migration in 3d microenvironments[END_REF][START_REF] Dozynkiewicz | Rab25 and clic3 collaborate to promote integrin recycling from late endosomes/lysosomes and drive cancer progression[END_REF]. RAB25 stably expressing cells display a signiicant increase in 5 1 integrin-dependent migration in 3D matrices containing ibronectin. An association between RAB25 and 1 integrin can be detected within invading pseudopodia. In addition, using cells expressing photoactivable paGFP-5 integrin and Cherry-RAB25 in combination with time-lapse microscopy, Norman and colleagues have demonstrated that the pool of 5 1 integrin at RAB25 vesicles is highly dynamic, and continuously delivered to the plasma membrane at pseudopodial tips. Interestingly, elevated expression of RAB25 leads to the retention of a pool of integrin at the pseudopodial tips [START_REF] Caswell | Rab25 associates with alpha5beta1 integrin to promote invasive migration in 3d microenvironments[END_REF]. Subsequent study has revealed that increased recycling of 5 1 integrin also requires the presence of RAB11FIP1 at the tips of invading pseudopods [START_REF] Caswell | Rab-coupling protein coordinates recycling of alpha5beta1 integrin and egfr1 to promote cell migration in 3d microenvironments[END_REF]. In a recent work of the same group, RAB25 was shown, in coordination with CLIC3, to promote the recycling of integrins from late endosomes/lysosomes to the plasma membrane. They proposed that in tumors lacking CLIC3, RAB25 will sort active integrins to lysosomes, thus attenuating integrin signaling. Conversely, in tumors with upregulated RAB25 and CLIC3, active integrins escape the degradation pathway and recycle back to the plasma membrane, thus enabling their signaling to drive tumor progression [START_REF] Dozynkiewicz | Rab25 and clic3 collaborate to promote integrin recycling from late endosomes/lysosomes and drive cancer progression[END_REF]. Taken together, these results revealed that RAB25 contributes to the tumorigenesis by promoting the traicking of integrins, key players in tumor invasion and metastasis.

When cancer cells invade in hypoxic 3D environments, they adapt to generate energy in oxygen independent ways and they express a range of genes involved in angiogenesis, cell survival, invasion and tumor progression, genes that are mostly regulated by the hypoxia inducible factors (HIF-1 and 1 ). In hypoxic conditions, HIF-1 enters the nucleus, binds to HIF-1 and triggers the expression of HIF1 target genes [START_REF] Gomez-Roman | Hypoxia-inducible factor 1 alpha is required for the tumourigenic and aggressive phenotype associated with rab25 expression in ovarian cancer[END_REF]. Ovarian cancer cell lines stably expressing RAB25 induce, in an oxygen-independent way, the expresion of HIF-1 which is found located in the nucleus. This HIF-1 induction by RAB25 was proposed to occur via ErbB2 regulation as well as ERK1/2 and mTOR signaling pathways.

RAB25 and chemotherapy-resistance. RAB25 was identiied in an ovarian cancer subgroup resistant to chemotherapy [START_REF] Cheng | The rab25 small gtpase determines aggressiveness of ovarian and breast cancers[END_REF][START_REF] Gomez-Roman | Hypoxia-inducible factor 1 alpha is required for the tumourigenic and aggressive phenotype associated with rab25 expression in ovarian cancer[END_REF]. In addition, it is suggested that chemoresistance induced by RAB25 is through HIF-1-dependent activation [START_REF] Gomez-Roman | Hypoxia-inducible factor 1 alpha is required for the tumourigenic and aggressive phenotype associated with rab25 expression in ovarian cancer[END_REF].

Bladder cancer is a major health issue worldwide that causes a considerable morbidity, mortality and overall health care costs due to frequent recurrences and inefective therapeutics. It is, therefore, essential to better understand the molecular mechanisms underlying bladder tumorigenesis to develop targeted therapies. FGFR3 is a promising candidate owing to the fact that FGFR3 alteration has been identiied as one of the most frequent events in bladder cancer, in particular in non-invasive tumors. FGFR3 is frequently activated by point mutation, translocation and overexpression. Although aberrant FGFR3s display oncogenic properties in bladder cancer, it remains partly characterized how FGFR3 activates the downstream signaling pathways, and whether there is a diference in the downstream signaling induced by the diferent aberrant forms of FGFR3.

As shown in Chapter 3, numerous evidence supports the view that traicking of a receptor tyrosine kinase may afect its signaling. To our knowledge, no study has addressed the relationship between FGFR3 traicking and signaling in the context of cancer, and in particular of bladder cancer. RAB25, along with closely related RAB11A and RAB11B known as key players of endocytic recycling process, is found to be upregulated or downregulated in a number of cancer screens in diferent studies. In addition, depending on the context, RAB25 has been found to display either protumorigenic or tumor suppressor properties. Gene expression data obtained in our laboratory or from a publicly available source indicate that RAB25 expression is signiicant higher in bladder tumors associated with altered FGFR3 compared to tumors without FGFR3 alterations. This suggests that RAB25 can play a protumorigenic role in tumors carrying altered FGFR3, potentially through an efect on the traicking of the receptor.

The Thesis project aims to investigate the role of endocytosis/endocytic recycling of FGFR3 in its signaling during bladder tumorigenesis.

Speciic objectives were designed: 1. Elucidate the role of RAB25, RAB11A, RAB11B and two of their efectors, RAB11FIP2 and MYO5B, in bladder cancer cells presenting FGFR3 alterations.

2. Compare the traicking and signaling of wild type and mutant S249C forms of FGFR3, and study the role of RAB11 and RAB25 in these processes.

3. Identify interaction partners of FGFR3 by mass spectrometry.

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III.

Results

Introduction

FGFR3 along with closely related FGFR1, FGFR2 and FGFR4 constitute a subfamily of the receptor tyrosine kinase (RTK) family [START_REF] Lemmon | Cell signaling by receptor tyrosine kinases[END_REF]. FGFR3 has three extracellular Immunoglobulin-like domains (I, II, III), a single transmembrane domain and an intracellular domain that contains a split tyrosine kinase domain. The alternative splicing of the third Immunoglobulin-like domain produces 2 isoforms of FGFR3 (FGFR3b and FGFR3c). FGFR3b is mainly expressed in epithelial cell populations while the FGFR3c isoform is present in non-epithelial cells [START_REF] Ornitz | Fgf signaling in the developing endochondral skeleton[END_REF]. FGFR3 activities promote diferent processes including proliferation, diferentiation, survival and migration. An important remark is that the functional outcome of FGFR3 signaling depends on the cellular context. FGFR3 acts as a physiological negative regulator of skeletal growth via inhibition of chondrocyte proliferation and the activating mutations of FGFR3 enhances the inhibition of cell growth in this cell type [START_REF] Ornitz | Fgf signaling in the developing endochondral skeleton[END_REF]. However, the activating mutations of FGFR3, even the same point mutation, cause excessive cell proliferation in epithelial cells [START_REF] Logié | Activating mutations of the tyrosine kinase receptor fgfr3 are associated with benign skin tumors in mice and humans[END_REF][START_REF] Bernard-Pierrot | Oncogenic properties of the mutated forms of ibroblast growth factor receptor 3b[END_REF].

From the irst discovery of four FGFR3 mutations in bladder and cervix cancers [START_REF] Cappellen | Frequent activating mutations of fgfr3 in human bladder and cervix carcinomas[END_REF], other FGFR3 mutations have been identiied and these mutations were found in various types of cancer such as multiple myeloma, lung cancer, prostate cancer and melanoma [START_REF] Turner | Fibroblast growth factor signalling: from development to cancer[END_REF][START_REF] Cerami | The cbio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data[END_REF]. However, high frequency of FGFR3 mutations seem to be restricted to bladder carcinomas, 50% in non-invasive muscle tumors and 10-15% in muscle-invasive tumors [START_REF] Neuzillet | A meta-analysis of the relationship between fgfr3 and tp53 mutations in bladder cancer[END_REF]; and to benign epidermal tumors, such as seborrheic keratosis (40-80% of samples tested) [START_REF] Logié | Activating mutations of the tyrosine kinase receptor fgfr3 are associated with benign skin tumors in mice and humans[END_REF][START_REF] Hafner | Mosaicism of activating fgfr3 mutations in human skin causes epidermal nevi[END_REF] as well as to epidermal nevi [START_REF] Hafner | Mosaicism of activating fgfr3 mutations in human skin causes epidermal nevi[END_REF][START_REF] Hernández | Fibroblast growth factor receptor 3 mutations in epidermal nevi and associated low grade bladder tumors[END_REF]. Recently, FGFR3 translocations leading to FGFR3-gene fusions, including FGFR3-TACC3 and FGFR3-BAIAP2L1 have been identiied in bladder carcinoma but also in glioblastoma and lung cancer [START_REF] Singh | Transforming fusions of fgfr and tacc genes in human glioblastoma[END_REF][START_REF] Wu | Identiication of targetable fgfr gene fusions in diverse cancers[END_REF]. Both mutations and translocations result in constitutively activated FGFR3 that displays oncogenic properties in bladder cancer [START_REF] Bernard-Pierrot | Oncogenic properties of the mutated forms of ibroblast growth factor receptor 3b[END_REF][START_REF] Di Martino | Mutant ibroblast growth factor receptor 3 induces intracellular signaling and cellular transformation in a cell type-and mutation-speciic manner[END_REF][START_REF] Williams | Oncogenic fgfr3 gene fusions in bladder cancer[END_REF]. However, how FGFR3 activates the downstream signaling pathways remains poorly understood. Thus, it is necessary to better understand the molecular mechanisms underlying FGFR3 signaling to develop a FGFR3-targeted therapy and treatment strategies for bladder cancer. This cancer is a major health issue worldwide that causes a considerable morbidity, mortality and overall health care costs due to frequent recurrences and inefective therapeutics [START_REF] Torre | Global cancer statistics[END_REF].

FGFRs as well as other RTKs transfer the extracellular stimuli to the cellular interior through activating several signaling cascades that inally results in physiological responses. In the classical model of signal transduction, transmembrane receptors can recruit many downstream substrates to the plasma membrane to activate signaling pathways, and endocytosis serves as receptor clearance pathway to attenuate receptor signaling. However, growing evidence supports the view that traicking of a RTK may afect its signaling [START_REF] Sorkin | Endocytosis and signalling: intertwining molecular networks[END_REF][START_REF] Miaczynska | Efects of membrane traicking on signaling by receptor tyrosine kinases[END_REF]. To date, no study has addressed the relationship between FGFR3 traicking and signaling in the context of cancer, and in particular of bladder cancer.

Endosomal traicking is spatiotemporally controlled by small RAB GTPases, which constitute the largest family of small GTPases with 70 members in human [START_REF] Kelly | The rab family of proteins: 25 years on[END_REF]. The traic of cargoes to the endocytic recycling compartment and from there to the plasma membrane is regulated by the RAB11 GTPase subfamily. This subfamily is composed of three proteins (RAB11A, RAB11B, RAB25) encoded by three distinct genes which share a high sequence homology [START_REF] Kelly | Rab11 proteins in health and disease[END_REF]. RAB11 proteins serve as regulators of endocytic recycling of many cell surface receptors and of endosomal membrane transport during cytokinesis [START_REF] Kelly | Rab11 proteins in health and disease[END_REF][START_REF] Schiel | Membrane dynamics during cytokinesis[END_REF]. To execute these functions, RAB11 proteins recruit motor adaptors directly or indirectly via RAB11 family-interacting proteins (RAB11FIPs). The best studied RAB11-RABFIPs-motor protein complex is the complex made by RAB11 with RAB11FIP2 and myosin Vb (MYO5B). This complex has been implicated in a wide range of endosomal recycling processes, for example recycling of transferrin receptor and integrins [START_REF] Welz | Orchestration of cell surface proteins by rab11[END_REF].

Alterations in RAB functions have been reported to be directly or indirectly responsible for many diseases, in particular, for cancers [START_REF] Stenmark | Rab gtpases as coordinators of vesicle traic[END_REF][START_REF] Chia | Emerging roles for rab family gtpases in human cancer[END_REF]. Among RAB GTPases involved in cancers, RAB25 is the protein whose association with tumor progression is the best known [START_REF] Mitra | Rab25 in cancer: a brief update[END_REF][START_REF] Agarwal | The emerging role of the rab25 small gtpase in cancer[END_REF]. Depending on the cellular context, RAB25 has been found to have either protumorigenic or tumor suppressor properties. The overexpression of RAB25 in ovarian cancer cells results in cell transformation in vitro and increases aggressiveness of cancer cells in vivo [START_REF] Cheng | The rab25 small gtpase determines aggressiveness of ovarian and breast cancers[END_REF][START_REF] Fan | Knockdown of rab25 expression by rnai inhibits growth of human epithelial ovarian cancer cells in vitro and in vivo[END_REF]. In contrast, in basal-and triple-negative breast tumor cells, overexpression of RAB25 decreases cell transformation properties such as, cell proliferation and anchorage-independent growth, and reduces tumor growth of xenografted breast carcinoma cells in nude mice [START_REF] Cheng | Loss of rab25 expression in breast cancer[END_REF][START_REF] Cheng | Tumor suppressor function of rab25 in triple-negative breast cancer[END_REF]. The mechanisms underlying the role of RAB25 in cell proliferation and apoptosis remain however poorly understood. On the other hand, several works have reported that RAB25 promotes tumor invasion and metastasis by regulating the endocytic recycling of integrins [START_REF] Caswell | Rab25 associates with alpha5beta1 integrin to promote invasive migration in 3d microenvironments[END_REF][START_REF] Dozynkiewicz | Rab25 and clic3 collaborate to promote integrin recycling from late endosomes/lysosomes and drive cancer progression[END_REF][START_REF] Tringali | The plasma membrane sialidase neu3 regulates the malignancy of renal carcinoma cells by controlling beta1 integrin internalization and recycling[END_REF].

In this study, we irst determined the status of genes coding for RAB and RABinteracting proteins in two groups of bladder cancers: tumors carrying mutated or translocated FGFR3, hereafter called altered FGFR3 tumors and tumors without these alterations, called non-altered FGFR3 tumors. Next, we investigated the role RAB25, RAB11A, RAB11B and two of their efectors, RAB11FIP2 and MYO5B, in bladder can-6.2 Results 69 cer cells carrying FGFR3 alterations by using siRNA depletion. We analyzed the efects of these depletions on cell viability, endocytic recycling, FGFR3 downstream signaling pathways and expression of FGFR3 target genes.

Results

6.2.1

RAB25 is upregulated in bladder tumos carrying altered FGFR3

In order to identify genes coding for RAB and RAB-interacting proteins associated with bladder cancer presenting alterations of FGFR3 (mutation and translocation genes), we performed a diferential expression analysis by using the LIMMA method between two groups: altered FGFR3 tumors and non-altered FGFR3 tumors. The list of 270 genes coding for RAB proteins and RAB-interacting proteins was established in a previous study [START_REF] Ho | Deregulation of rab and rab efector genes in bladder cancer[END_REF]. Two data sets were collected, one from our laboratory (HuExon dataset), constituting of 98 supericial tumors and 106 muscle invasive tumors (63 and 15 tumors carrying FGFR3 alteration, respectively), and the other from a publicly available source The Cancer Genome Atlas (TCGA dataset), consisting of 371 muscleinvasive tumors among which 50 tumors carry FGFR3 alteration. 42 genes out of 270 genes tested are diferentially expressed between the two groups of altered and nonaltered supericial tumors (Figure 6.1). For muscle-invasive tumors in the HuExon dataset and the TCGA dataset, we obtained 15 genes and 122 genes, respectively, differentially expressed (Figures 6.2, 6.3). The results indicated that RAB25 expression was signiicantly higher in altered FGFR3 tumors compared to non-altered FGFR3 tumors (Figure 6.4.A). In supericial tumors and muscle-invasive tumors of the HuExon dataset, the fold change (FC) was 1.2 and 1.7, respectively; in invasive tumors of the TCGA dataset, the FC was 2.3. RAB25 appeared twice as the RAB having the most upregulated expression in tumors presenting alterations of FGFR3 (Figures 6.1, 6.2, 6.3). Moreover, RAB11A and RAB11B are also signiicantly upregulated in the 50 altered-FGFR3 tumors of 371 muscle-invasive tumors of TCGA with FC of 1.5 and 1.2, respectively (Figure 6.4.B,C). On the other hand, there was no diference in the expression levels of RAB11 efectors including RAB11FIP2 and MYO5B in tumors presenting or not alterations of FGFR3 (Figure 6.4.D,E). Our indings suggest that in tumors carrying altered FGFR3, the altered endocytic recycling due to the upregulation of RAB25 and RAB11 can promote bladder progression.

6.2.2

Depletion of RAB25/RAB11 proteins and their efectors inhibits cell viability of altered FGFR3 expressing cells

In order to investigate the role of RAB25, RAB11A, RAB11B, RAB11FIP2 and MYO5B in bladder cancer, we examined the mRNA expression of these genes in normal human urothelium cells (NHU cells) and 6 human bladder cancer cell lines (Figure 6.5.A-E). The characteristics of these cancer cell lines is summarized in Figure 6 This table provides the results of the diferential expression analysis, performed with the LIMMA method, between supericial tumors associated with altered FGFR3 and without FGFR3 alterations from the TCGA data set. symbol, Gene symbol; adjusted P-value, adjusted p-value using Benjamini et Hochberg procedure; Foldchange, estimate the fold change tumors associated with altered FGFR3 versus without FGFR3 alterations.

Results 73

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • FC = 1.2 p<0.05
superficial HuExon data set -700 -500

-300

RAB25 expression

Altered (n=63)

Non-altered (n=35)

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • -200 -600 -400 -0 FC = 1.7 p<0.05 invasive HuExon data set RAB25 expression Altered (n=15) Non-altered (n=91) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Altered (n=50) -30k -20k -10k -0 Non-altered (n=321) FC = 2.3 p<0.01

RAB25 expression

TCGA data set

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • -20k -15k -10k -5k Altered (n=50)
Non-altered (n=321)

RAB11B expression

TCGA data set p<0.01

FC = 1.2 • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • -900 -700 -500 -300 RAB11A expression Altered (n=63) Non-altered (n=35)
superficial HuExon data set

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • -30k -20k -10k -40k p<0.0001 FC = 1.5 Altered (n=50) Non-altered (n=321) -0 RAB11A expression TCGA data set • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • -800 -600 -400 -200 -1000 Altered (n=63) Non-altered (n=35)
RAB11B expression superficial HuExon data set

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Altered (n=15) Non-altered (n=91) -500 -400 -300 -200

RAB11B expression

invasive HuExon data set

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • -800 -600 -400 -200 Altered (n=63) Non-altered (n=35)

RAB11FIP2 expression

superficial HuExon data set

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • invasive HuExon data set -200 -600 -400 Altered (n=15) Non-altered (n=91) RAB11FIP2 expression • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • -4k -2k -0 Altered (n=50) Non-altered (n=321) RAB11FIP2 expression TCGA data set • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • -200 -100

MYO5B expression

superficial HuExon data set Altered (n=63)

Non-altered (n=35)

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • -250 -200 -150 -100 -50 Altered (n=15) Non-altered (n=91)
invasive HuExon data set MYO5B expression

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • -20k -15k -10k -5k -0 MYO5B expression Altered (n=50) Non-altered (n=321)
TCGA data set the cancer cell lines (Figure 6.5.B-D). Interestingly, RAB25 and MYO5B mRNA levels displayed higher variability among the cell lines (Figure 6.5.A and E). Conirmed by Western blotting, the RAB25 protein level displayed variability among the cell lines tested (Figure 6.5.G). Therefore, to examine the role of these proteins in cell viability of bladder cancer cells carrying altered FGFR3, we selected two cell lines, MGHU3 and RT112 cells. Indeed, MGHU3 cells endogenously express FGFR3 with a point mutation (Y375C). RT112 cells show a high level of FGFR3 ampliication and upregulation of both wild type FGFR3 and the FGFR3-TACC3 fusion protein (Figure 6.5.F). Moreover, cell proliferation and transforming properties of these cell lines are dependent on constitutively activated FGFR3 [START_REF] Bernard-Pierrot | Oncogenic properties of the mutated forms of ibroblast growth factor receptor 3b[END_REF][START_REF] Williams | Oncogenic fgfr3 gene fusions in bladder cancer[END_REF]. We irst investigated the efects of RAB11A, RAB11B, RAB25, RAB11FIP2 and MYO5B depletion by siRNA on cell viability. For a given gene, we tested knockdown eiciency by RT-qPCR of three individual siRNAs targeting diferent sites of this gene. Then, one siRNA for each gene was selected for following experiments if the expression of target gene is reduced over 80% (data not shown). After 72h siRNA transfection, the viability of RT112 and MGHU3 cells was measured by MTT assays (Figure 6.7). This assay allows indirect measurement of cell viability after siRNA silencing or a drug treatment.

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Altered (n=15) Non-altered (n=91)
A B C F E D M G H U 3 R T
In parallel, the eicacy of siRNAs was evaluated by RT-qPCR or Western blot depending on the availability of antibodies (Figure 6.8). Compared to cells transfected with control siRNA, in MGHU3 cells, the inhibitory efect on cell viability ranged from 50% to 70% (Figure 6.7.A). Also, the viability was reduced to ∼50% in RT112 cells with all siRNAs (Figure 6.7.B). Because RAB25, RAB11A and RAB11B share a high sequence identity [START_REF] Kelly | Rab11 proteins in health and disease[END_REF], these proteins were simultaneously depleted by using a combination of siRNAs to test their functional redundancy (Figure 6.7.A,B). In both cell lines, the cell viability was decreased to the same level by either one or two or three siRNA against the RAB11 subfamily genes, indicating that RAB11A, RAB11B, RAB25 are necessary for the viability of RT112 and MGHU3 cells. In other words, the three members of the RAB11 subfamily have a distinct role in the cell viability. To verify whether this efect on cell viability is speciic for RAB25, RAB11A, RAB11B and their efectors RAB11FIP2 and MYO5B, we transfected RT112 cells with siRNA targeting other RAB GTPases, including RAB7A, RAB7B and RAB21. RAB7A and RAB7B localize to late endosomes and are involved in the degradation pathway [START_REF] Jordens | The rab7 efector protein rilp controls lysosomal transport by inducing the recruitment of dyneindynactin motors[END_REF]. RAB21 is implicated in transport events at the level of early endosomes [START_REF] Pellinen | Small gtpase rab21 regulates cell adhesion and controls endosomal traic of beta1-integrins[END_REF]. The efects on cell viability induced by depletion of RAB7 or RAB21 was ∼20%, lower than that

RAB11B level s iC tr l s iR A B 1 1 A + B + 2 5 s iR A B 1 1 A + B s iR A B 1 1 A + 2 5 s iR A B 1 1 B + 2 5 s iR A B 1 1 A s iR A B 1 1 B s iR

RAB11B level s iC tr l s iR A B 1 1 A + B + 2 5 s iR A B 1 1 A + B s iR A B 1 1 A + 2 5 s iR A B 1 1 B + 2 5 s iR A B 1 1 A s iR A B 1 1 B s iR

Results 77

observed after depletion of members of the RAB11 subfamily (Figure 6.7.C). Taken together, these results suggest that altering the endocytic recycling results in the inhibition of cell viability of cells displaying alterations of FGFR3.

To determine whether this efect is speciic to bladder cancer cells expressing FGFR3 or more general in diferent subgroups of bladder cancers, we next repeated the depletion experiments in several bladder cancer cell lines, including TCCSup, UMUC9, UMUC5 and HT1376 cell lines (Figure 6.9). These cell lines do not express FGFR3 (Figure 6.5.F). Knockdown eiciency was conirmed by RT-qPCR or Western blot (Figure 6.10). In these cell lines, the RAB11/RAB25 or MYO5B depletion had no efect or induced a lower inhibition in cell viability than in RT112 and MGHU3 cells (Figure 6.9). Of note, the siRNA targeting RAB11B had an important efect on HT1376 (∼80%), therefore, we examined this efect with another siRNA targeting RAB11B. The inhibition of cell viability was less important (20%) (data not shown), suggesting that the irst siRNA targeting RAB11B had an of-target in HT1376 cells. Taken together, these indings suggest that the inhibition of cell viability induced by the depletion of RAB11/RAB25 and their efectors is speciic in bladder cancer cells presenting alterations of FGFR3. 6.2 Results 79

6.2.3

Efects on cell viability of a combination of RAB11/RAB25 depletion with FGFR3 depletion or treatment with a FGFR inhibitor

Altered FGFR3 has been shown to display oncogenic properties in bladder cancer cells in vitro and in vivo [START_REF] Bernard-Pierrot | Oncogenic properties of the mutated forms of ibroblast growth factor receptor 3b[END_REF][START_REF] Di Martino | Mutant ibroblast growth factor receptor 3 induces intracellular signaling and cellular transformation in a cell type-and mutation-speciic manner[END_REF][START_REF] Williams | Oncogenic fgfr3 gene fusions in bladder cancer[END_REF]. Indeed, previous studies from our laboratory revealed that knockdown of FGFR3 in RT112 and MGHU3 cells inhibited cell viability. We therefore wanted to determine whether the impact on cell viability of RAB25/RAB11 silencing relects the role of FGFR3 in these cell lines. We transfected RT112 cells with a cocktail of siRNAs targeting FGFR3 and RAB25/RAB11. In cells transfected with only siRNA against FGFR3, the efect on cell viability was ∼80%, a value higher than that obtained following RAB25/RAB11 or MYO5B depletion (50% or 40%, respectively) (Figure 6.11.A). Of note, the simultaneous depletion of FGFR3 with either RAB25/RAB11 or MYO5B has the same efect (80% inhibition) compared to the depletion of FGFR3 alone. In parallel, we treated RT112 cells depleted for RAB25/RAB11 with either a FGFR inhibitor PD173074 (0.5 M) or DMSO (0.1%) as control for 24h.

The treatment with the FGFR inhibitor induced a dramatic reduction in cell viability and the number of surviving cells were about the same in RAB11/RAB25-depleted cells treated with the FGFR inhibitor (Figure 6.11.B). These results indicate that the depletion of RAB11/RAB25 is not suicient to induce an efect on cell viability as important as FGFR3 depletion does. The inhibition of RAB25/RAB11 or MYO5B however does not have an additive efect on the inhibition of cell viability by either a siRNA targeting FGFR3 or a tyrosine kinase inhibitor, suggesting that RAB25/RAB11 proteins could be involved in FGFR3 signaling. On the other hand, it is possible that the complex of endocytic recycling and FGFR3 signaling represent two separate pathways that are both required for cell viability.

6.2.4

FGFR3 localizes to TFRC-, RAB25-and RAB11A-positive vesicles

To date, the cellular localization of FGFR3 has not been characterized in the context of bladder cancer. We therefore addressed this question by using RT112 cells transiently expressing FGFR3-GFP due to the lack of antibodies speciically recognizing endogenous FGFR3 by immunoluorescence staining. We generated the C-terminal GFP tagged FGFR3 for the wild type form (FGFR3-GFP). FGFR3-GFP was shown by co-immunoprecipitation to interact with HSP90, a well-known partner of FGFR3 [START_REF] Laederich | Fibroblast growth factor receptor 3 (fgfr3) is a strong heat shock protein 90 (hsp90) client: implications for therapeutic manipulation[END_REF], suggesting that FGFR3-GFP is fully functional in RT112 cells (data not shown). 24h after transfection, RT112 cells were ixed and the subcellular distribution of FGFR3 was analyzed using 3D deconvolution microscopy. FGFR3 displayed a characteristic punctuate, vesicular pattern throughout the cytoplasm (Figure 6.12.B). Fluorescent signal was weakly detected on the plasma membrane probably due to limitations of wideield light microscopy.

We then used various antibodies against marker proteins of endosomal compartments, including anti-TFRC (transferrin receptor), anti-EEA1, anti-LAMP1, anti-CD63 and anti-RAB25 to further identify the nature of FGFR3 punctuate structures (Figure 6.12.A). No diference in the subcellular localization of these markers was observed between non-transfected RT112 cells and cells transfected with FGFR3-GFP (data not shown). We quantiied the colocalization between two structures containing FGFR3 and these markers by using the plugin JACOP in ImageJ software to calculate Manders' overlap coeicients [START_REF] Manders | Measurement of co-localization of objects in dual-colour confocal images[END_REF][START_REF] Bolte | A guided tour into subcellular colocalization analysis in light microscopy[END_REF] (Figure 6.12.C). TFRC localizes mainly to recycling endosomes at steady state. A signiicant degree of colocalization of TFRC with FGFR3 (Manders' coeicient=0.18) was observed in punctuate structures throughout the cytoplasm. A similar degree of colocalization was observed with RAB25 (Manders' coeicient=0.2). Immunolabelling of the early endosomes with EEA1 indicated that FGFR3 was found in the early endosomal compartment (Manders' coeicient=0.15). There was a small fraction of FGFR3 structures which colocalized with LAMP1 or CD63, markers of late endosomes and lysosomes (Manders' coeicient =0.1). Of note, the degree of colocalization between FGFR3 and RAB25 or TFRC was signiicant higher than that of LAMP1 or CD63. In addition, we co-transfected RT112 cells with FGFR3-GFP and RAB11A-mcherry, and quantiied the colocalization between FGFR3 and RAB11A. As for RAB25, 25% of FGFR3-positive structures colocalized with RAB11A (Figure 6.12.A,C). Thus, these results indicate that FGFR3 resides in diferent intracellular compartments but preferentially localizes to TFRC-, RAB25-, RAB11A-positive recycling compartments.

        siMYO5B                                                      A   ns     siCtrl siRAB11s siFGFR3 + + + + + + + + + + -- -- -- - - - - - - - - -- - - B -- --

6.2.5

RAB25 and RAB11 depletion alters the subcellular distribution of FGFR3

To investigate the role of RAB25/RAB11 on the subcellular localization of FGFR3, RT112 cells were transfected with control or a cocktail of RAB11A, RAB11B and RAB25targeting siRNAs for 48h, followed by FGFR3-GFP transfection for 24h. Knockdown eiciency was conirmed by RT-qPCR and Western Blot. In control RT112 cells, TFRC- and FGFR3-positive vesicles were both distributed throughout the cytoplasm. In RAB25and RAB11-depleted cells, we observed tubular TFRC-positive structures (Figures 6.13), as previously reported in other cell lines [START_REF] Ullrich | Rab11 regulates recycling through the pericentriolar recycling endosome[END_REF][START_REF] Haugsten | Photoactivation approaches reveal a role for rab11 in fgfr4 recycling and signalling[END_REF]. In these cells, FGFR3-positive structures appeared larger and more concentrated in the perinuclear region than in controls. In addition, the extent of colocalization with TFRC was signiicantly increased (28% versus 18% in controls) (Figure 6.14). Of note, colocalization of FGFR3 with CD63 or LAMP1 was also signiicantly increased (Figures 6.13 and 6.14) but not that with EEA1. These data suggest that RAB11/RAB25 proteins play a role in the recycling of FGFR3 and that the inhibition of recycling favors its degradation.

6.2.6

Depletion of RAB25/RAB11 has diferential efects on the known signaling pathways of FGFR3

RAB25/RAB11 depletion leading to altered recycling of FGFR3, we tested whether this can potentially afect its signaling. We analyzed activation of downstream substrates of FGFR3 in RAB25/RAB11-depleted RT112 cells. Previous studies in our laboratory demonstrated that PI3K-AKT, P38 and ERK1/2 are three main signaling pathways activated by altered FGFR3 in bladder cancer [START_REF] Bernard-Pierrot | Oncogenic properties of the mutated forms of ibroblast growth factor receptor 3b[END_REF][START_REF] Mahe | Caractérisation des voies de signalisation des oncogènes FGFR3 muté et FGFR3-TACC3 dans les carcinomes de vessie[END_REF]. Interestingly, elevated level of phosphorylated P38 was observed following RAB25 depletion. P-AKT was increased only when cells were depleted for RAB11A or RAB11B or RAB25. RAB11B depletion induced an increase in phosphorylation of ERK1/2 (Figure 6.15.A). We then investigated whether the diferential efects on downstream signaling substrates relect the altered activity of FGFR3 in these pathways. The P38 as well as the AKT pathway were shown to play a more important role than the ERK1/2 pathway in the proliferation of RT112 cells. We determined the phosphorylation of P38 after simultaneously depleting RAB25 and FGFR3 or after FGFR inhibitor PD173074 treatment in RAB25-depleted cells (as described in the experiments on cell viability). The results indicate that the depletion of FGFR3 alone reduced the phosphorylation levels of P38, in contrast to the depletion of RAB25 alone (Figure 6.15.B). The level of P-P38 after simultaneous depletion for FGFR3 and RAB25 was slightly reduced compared to control. In cells depleted for RAB25 and treated with PD173074, P-P38 was higher than in control cells, maybe due to the fact that PD173074 did not completely inhibit P-P38.

RT112 l i p o s i C t r l s i C t r l 2 0 n M s i C t r l 3 0 n M s i R A B 1 1 A s i R A B 1 1 A + B s i R A B 1 1 A + 2 5 s i R A B 1 1 B + 2 5 s i R A B 1 1 A + B + 2 5 s i R A B 1 1 B s i R A B 2 5 s i C t r l s i C t r l s i C t r l s i F G F R 3 s i R A B 2 5 + F G F R 3 s i R A B 2 5 s i R A B 2 5 s i R A B

6.2.7

RAB25/RAB11 depletion has not the same efect compared to FGFR3 depletion on the expression of FGFR3 target genes

To further unravel the origin of the inhibitory efects on cell viability observed after knockdown of RAB11 and their efectors in cells carrying altered FGFR3, we tested whether the expression of FGFR3 target genes was afected. FGFR3 target genes were identiied in a separate study performed in our laboratory. Briely, we compared the transcriptome of the RT112 and MGHU3 cell lines treated with two or three siR-NAs targeting FGFR3 or with the transfection reagent (Lipofectamine) as control. We

Results 85

found that 414 genes were signiicantly deregulated by siRNA treatment (283 genes downregulated and 131 genes upregulated compared to control). Among deregulated genes, we selected three genes which have a potential function in FGFR3 activity including DUSP6, TIMP2 and GATA3. DUSP6 is a well-known negative regulator of FGFR signaling via the inhibition of the ERK1/2 pathway [START_REF] Ekerot | Negativefeedback regulation of fgf signalling by dusp6/mkp-3 is driven by erk1/2 and mediated by ets factor binding to a conserved site within the dusp6/mkp-3 gene promoter[END_REF]; TIMP2 is an inhibitor of metalloproteinases [START_REF] Seo | Timp-2 mediated inhibition of angiogenesis: an mmp-independent mechanism[END_REF]; GATA3 serves as a transcription factor for diferentiation [START_REF] Chou | Gata3 in development and cancer diferentiation: cells gata have it![END_REF]. We also selected MYC since this gene was previously shown in our laboratory to be inhibited following FGFR3 depletion in RT112 and MGHU3 cells. After 72h transfection with siRNA targeting RA25B/RAB11 or a cocktail of siRNA targeting these RABs and FGFR3 in RT112 cells, we performed mRNA extractions and reverse transcription-PCR. Then, the expression of the four genes described above was determined by RT-qPCR, normalized to TBP gene expression. The results indicated that the single, double or triple depletion of RAB11A/RAB11B/RAB25 induced a ∼40-50% inhibition of MYC expression compared to control siRNA. However, in FGFR3depleted cells or in cells depleted for both FGFR3 and RAB11/RAB25, MYC expression was inhibited up to 80% (Figure 6.16.A). This efect of FGFR3 on MYC expression is consistent with other studies in our laboratory. In the case of DUSP6, its expression was also reduced up to ∼80% when cells were knockdown for FGFR3 alone or for FGFR3 and RAB11/RAB25 (Figure 6.16.B). RAB11/RAB25 depletion only moderately afected DUSP6 expression. We next examined the expression of GATA3 and TIMP2 that are upregulated following FGFR3 depletion in microarray data (Foldchange = 1.7 and 2.6, respectively). By RT-qPCR, we obtained the same result, FGFR3 silencing increasing three times the expression of GATA3 (Figure 6.16.C). GATA3 was upregulated by 150% compared to control siRNA when RAB11A was depleted but reduced by 50% in RAB11/RAB25-knockdown cells. The upregulation of GATA3 after simultaneous depletion of RAB11 and RAB25 and FGFR3 was less pronounced than in the case of FGFR3 depletion alone but still two times compared to control siRNA. FGFR3 but not RAB11 depletion induced a massive upregulation of TIMP2 (Figure 6.16.D). Taken together, these results indicated that RAB11 depletion has less efect than FGFR3 depletion does on the expression of several FGFR3 target genes.

A B D C

s i C t r l s i R A B 1 1 A + B + 2 5 s i R A B 1 1 A + B s i R A B 1 1 A + 2 5 s i R A B 1 1 B + 2 5 s i R A B 1 1 A s i R A B 1 1 B s i R A B 2 5 s i F G F R 3 s i F G F R 3 + 3 s i R A B 0 50 100 150

MYC level normalized to TBP expression level (% to control)

s i C t r l s i R A B 1 1 A + B + 2 5 s i R A B 1 1 A + B s i R A B 1 1 A + 2 5 s i R A B 1 1 B + 2 5 s i R A B 1 1 A s i R A B 1 1 B s i R A B 2 5 s i F G F R 3 s i F G F R 3 + 3 s i R A B 0
s i R A B 1 1 A + B + 2 5 s i R A B 1 1 A + B s i R A B 1 1 A + 2 5 s i R A B 1 1 B + 2 5 s i R A B 1 1 A s i R A B 1 1 B s i R A B 2 5 s i F G F R 3 s i F G F R 3 + 3 s i R A B 0
s i R A B 1 1 A + B + 2 5 s i R A B 1 1 A + B s i R A B 1 1 A + 2 5 s i R A B 1 1 B + 2 5 s i R A B 1 1 A s i R A B 1 1 B s i R A B 2 5 s i F G F R 3 s i F G F R 3 + 3 s i R A B 0

6.2.8

Dyngo4a treatment increases the phosphorylation of AKT and ERK1/2 but not that of P38

In order to investigate the potential role of endocytosis in FGFR3 signaling, we treated RT112 cells with Dyngo4a. Dyngo4a is a potent dynamin inhibitor which acts at an allosteric site in the G domain of dynamin, resulting in blocking dynamin-dependent endocytosis [START_REF] Mccluskey | Building a better dynasore: The dyngo compounds potently inhibit dynamin and endocytosis[END_REF]. We irst determined the minimum concentration of Dyngo4a required to block endocytosis by testing a range of Dyngo4a concentrations. The impact on transferrin internalization and cell morphology were taken into account. 60 M appeared as a good concentration because it did not afect the cell morphology and transferrin internalization was blocked at this concentration (Figure 6.17.A). In RT112 cells, although we could not verify whether FGFR3 internalization was efectively blocked due to the lack of FGFR3 antibodies working suitable for immunoluorescence staining. The fact that transferrin was accumulated at the plasma membrane after 30 min treatment with Dyngo4a suggests other receptors including FGFR3 were likely blocked at the plasma membrane.

Following treatment with 60 M Dyngo4a or DMSO 0.1% for 15 min, 30 min, 1h or 2h, RT112 cells were lysed and protein levels were determined by Western blotting. The results indicate an increased phosphorylation of AKT and ERK1/2 but not that of P38 (Figure 6.17.B-D). In RT112 cells, AKT, ERK and P38 pathways are mainly activated by FGFR3 activity independently of ligand stimulation [START_REF] Bernard-Pierrot | Oncogenic properties of the mutated forms of ibroblast growth factor receptor 3b[END_REF][START_REF] Mahe | Caractérisation des voies de signalisation des oncogènes FGFR3 muté et FGFR3-TACC3 dans les carcinomes de vessie[END_REF]. These indings suggest that P-38 can be activated by FGFR3 at either the plasma membrane or intracellular compartments. In contrast, the increased phosphorylation of AKT and ERK1/2 observed following Dyngo4a treatment is probably due to the accumulation of FGFR3 at the plasma membrane, suggesting that FGFR3 phosphorylates AKT and ERK1/2 at the plasma membrane.

6.3

Materials and methods

Microarray data

Transcriptomic data from 204 human bladder samples and seven cell lines were obtained using Afymetrix Human Exon 1.0 ST DNA Array. RNA ampliication, cDNA probe labeling and hybridization were performed as described on the Afymetrix website. Raw data were normalized by the robust multiarray average (RMA) method available in the R package afy. The annotation used is based on the custom CDF iles from BrainArray [START_REF] Dai | Evolving gene/transcript deinitions signiicantly alter the interpretation of genechip data[END_REF], Ensembl Gene version 17. The Limma of R package from Bioconductor [START_REF] Smyth | Linear models and empirical bayes methods for assessing diferential expression in microarray experiments[END_REF] was used to identify genes diferentially expressed in FGFR3 altered tumors compared to non-altered tumors. Clinical and RNA-seq data related to 371 muscleinvasive bladder tumor samples were collected from The Cancer Genome Atlas (Provisional data 2015) [205]. Raw data was normalized by the trimmed mean of M values (TMM) method with the R package [START_REF] Robinson | A scaling normalization method for diferential expression analysis of rna-seq data[END_REF]. Diferential expression was analyzed with the voom Limma in the R package from Bioconductor [START_REF] Law | Precision weights unlock linear model analysis tools for rna-seq read counts[END_REF][START_REF] Smyth | Linear models and empirical bayes methods for assessing diferential expression in microarray experiments[END_REF]. For each diferential expression analysis, the p-values were adjusted for multiple testing by Benjamini et Hochber FDR methods. Genes with an adjusted p-value below 0.05 were considered to be diferentially expressed.

Cell culture and reagents

Bladder cancer cell lines used in this study (UMUC5, UMUC9, HT1376) were purchased from ECACC and TCCSup cells from ATCC. The MGHU3 cell line was a gift from Yves Fradet (CRC, Quebec). All cell lines were maintained under 5% CO2 at 37 ∘ C in Dulbecco's modiied Eagle's medium (DMEM) (Gibco, Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Lonza), except for the RT112 cells cultured in RPMI (Gibco, Life Technologies) with 10% FBS. DMSO, Tyrosine kinase FGFR inhibitor PD173074 (P2499) was purchased from Sigma. Dyngo4a (ab120689) was purchase from Abcam. DAPI (D1306) was purchase from Molecular Probes.

Antibodies

Antibodies were purchased from Sigma Aldrich: anti-FGFR3 (F0425), anti-alpha-tubulin (T6199); from Abcam: anti-FGFR3 (ab133644), anti-CD63 (ab23792); from Cell Signaling Technology: anti-RAB25 (4314); anti-RAB11A (2413); anti-RAB7A/B (9367); anti-p44/42 MAPK (9102); anti-phospho-p44/42 MAPK (Thr202/Tyr204) (9101); anti-AKT (9272); anti-phospho-AKT Ser473 (4060); anti-P38 MAPK (9212); anti-phospho-P38 MAPK (Thr180/Tyr182) (4511); anti-mouse HRP-linked (7076); anti-rabbit HRPlinked (7074); from Santa Cruz Biotechnology: EEA1 antibodies (sc-6414); from BD Pharmingen: anti-LAMP1 CD107a (553792); from Invitrogen: Goat anti-mouse IgG Alexa Fluor 594 (a11032), Goat anti-rabbit IgG Alexa Fluor 594 (a11037), anti-Transferrin (136800); from Jackson ImmunoResearch: Donkey anti-goat IgG Cy3. Anti-RAB21 was a kind gift from Johanna Ivaska's laboratory.

Plasmid constructs and transfection

The FGFR3-GFP construct was generated by subcloning the HindIII-BamHI fragment from pcDNA3.1 containing wild type FGFR3 into pEGFP-N3 (Clontech). Full length human wt RAB11A lanked with attB1.1 and attB2.1 were synthesized and inserted into pDONR207 (BP cloning reaction, Invitrogen), then transfered into Gateway compatible versions of pmCherry-C1 (Clontech) (LR cloning reaction, Invitrogen). All constructs were sequence-veriied by Sanger sequencing. RT112 cells were transfected with this construct using Fugene HD Transfection Reagent (Promega) according to the manufacturer's instructions.

RNA interference

siRNAs were transfected with Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer's instructions, and cultured for 72h for further manipulations. Depletion eiciency was estimated by RT-qPCR and/or Western blotting (≥80% inhibition). All siRNAs were purchased from Qiagen, except for FGFR3 (Ambion) and RAB21 (L-009450-00, Dharmacon). The target sequences of siRNA against RAB7A no5: CACGTAGGCCTTCAACACAAT and siRNA against RAB7B no5: AAGGTGGACCT-GAAACTCATT. List of siRNA sequences and concentration used:

MTT assay

After siRNA transfection or treatment with inhibitor (time as indicated in igures), medium was changed, MTT (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) (Sigma) was added to the cells to a inal concentration of 1 mg/mL. After 30 min incubation at 37 ∘ C in a 5% CO2, the purple formazan was solubilized in DMSO and quantiied. Absorbance was measured at 550 nm using a microplate reader. For MTT assay, each condition was performed in triplicate wells.

Real-time quantitative PCR

RNA was extracted from cells with RNAeasy kits (Quiagen). One microgram of total RNA was reverse-transcribed with the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). Predesigned assay was used to quantify human TBP gene expression (Applied Biosystems) and custom assays were used for the other genes (see table ). For custom assays, primers and probes were designed with Probe Finder software via the Universal Probe Library Assay Design Center (Roche). RT-qPCR was carried out with the LightCycler 480 Instrument (Roche) in a 20 l reaction mixture containing 10-20 ng of reverse-transcribed RNA, 1x LightCycler 480 Probe Master, 25 M each of the forward and reverse primers and 10 M of the UPL probe (or 1x the predesigned assay probe). All expression assays were run in the same thermal cycling conditions, including an initial step at 95 ∘ C (10 min), followed by 40 cycles at 95 ∘ C (10 s), 60 ∘ C (30 s) and 72 ∘ C (10 s). For each gene, the amount of mRNA was normalized to that of the TBP gene by the 2 -∆∆ method.

Western blotting

Protein extracts were obtained using lysis bufer (50mM Tris-HCl (pH 6.8), 2% SDS, 5% glycerol, DTT 2mM, 2.5 mM EDTA, 2.5 mM EGTA) freshly supplemented with Protease and phosphatase inhibitor (Roche)). Cell lysates were clariied by centrifugation. Protein concentration was determined with a BCA Protein Assay-Reducing Agent Compatible kit (ThermoFischer). Proteins (8 g to 20 g) were separated by SDS-PAGE using 4-15% precast Tris-glycine gel (Biorad) and transferred to nitrocellulose membranes using Trans-Blot Turbo transfer system (Biorad). Membranes were then blocked with 5% non-fat milk or 5% BSA at room temperature. Membranes were then incubated with the primary antibody overnight at 4 ∘ C and the secondary antibody for 45 min at room temperature. Signals were detected with the Clarity Western ECL (BioRad), Western blots were analyzed with a Fujiilm LAS-3000 imager or with ChemiDoc MP imaging systems using Imagelab software (Biorad).

Immunoluorescence staining

Cells were ixed for 15 min with 4% PFA in PBS at room temperature. After permeabilization with 0.5% saponin (Sigma-Aldrich) and 2% BSA (Sigma-Aldrich), cells were incubated with primary and secondary antibodies before being mounted with

Materials and methods 91

Mowiol (Biovalley). Fixed samples were imaged with an upright wideield microscope from Leica equipped with 100X CFIPlanAPo VC oil immersion objective (Na 1.4 CFI), a piezoelectric motor (Physik Instrumente) and highly sensitive cooled interlined charge-coupled device (CCD) camera (CoolSNAP HQ2, Photometrics). Z-dimension series of images were taken every 0.2 m by Metamorph software (Molecular Devices).

After deconvolution (Meinel algorithm, [START_REF] Sibarita | Deconvolution microscopy[END_REF]), colocalization between structure was analyzed with ImageJ 1.50 software [START_REF] Schneider | Nih image to imagej: 25 years of image analysis[END_REF]. Manders' overlap coeicients were calculated using the JACOP plugin for ImageJ [START_REF] Bolte | A guided tour into subcellular colocalization analysis in light microscopy[END_REF].

Transferrin internalization

For transferrin internalization experiment, RT112 cells were starved for 4h in serum free medium and incubate with transferrin-A555 (10g/ml) (Invitrogen) for 45 min at 4 ∘ C. Cells were then washed with cold PBS and chased at 37 ∘ C with serum free medium for 15 min. Cells were ixed with 4% PFA in PBS and mounted with Mowiol.

Statistical analysis

All functional experiments were carried out twice or three times, in triplicate. Data are expressed as means ± SEM. Student's t-tests were used for the statistical analysis.

The control siRNA group or the DMSO group was used as the reference group.

Traicking of wild type and mutated S249C FGFR3 7

Introduction

FGFR3 mutations in bladder cancer were irst reported by Radvanyi's group over ifteen years ago [START_REF] Cappellen | Frequent activating mutations of fgfr3 in human bladder and cervix carcinomas[END_REF]. They found single nucleotide substitutions of FGFR3 in 9 of 26 bladder carcinomas (35%). Since then, 11 diferent mutations have been identiied in urothelial tumors. The most frequent mutations are S249C (66.6%), Y375C (15.1%), R248C (9.7%) and G372C (4.3%) [START_REF] Goebell | Bladder cancer or bladder cancers? genetically distinct malignant conditions of the urothelium[END_REF].

In this study, we irst generated plasmid encoding the wild type (WT) and S249C FGFR3 tagged with GFP at the C-terminus. Next, we compared the subcellular distribution of these two forms in HeLa cells transiently expressing WT or S249C FGFR3-GFP. Finally, we evaluated the efects of RAB11A/B depletion by siRNA on the subcellular localization and signaling of the two forms of FGFR3.

Results

7.2.1

Generation of WT and mutant S249C FGFR3-GFP expressing cells

The cellular distribution of wild type (WT) and mutant S249C forms of FGFR3 was studied in HeLa cells transiently transfected with diferent FGFR3 constructs. HeLa cells do not express detectable levels of endogenous FGFR3 [START_REF] Haugsten | Diferent intracellular traicking of fgf1 endocytosed by the four homologous fgf receptors[END_REF]. This was also conirmed by Western blotting (Figure 7.1.A). We generated the plasmids encoding FGFR3 tagged with GFP at the C-terminus or N-terminus for each form. 24h after transfection with these constructs, HeLa cells were ixed and analyzed by luorescence microscopy. The N-terminal GFP-tagged constructs of the two forms of FGFR3 localized to the endoplasmic reticulum, suggesting that GFP-FGFR3s may not be correctly folded to exit the endoplasmic reticulum (data not shown). In contrast, the C-terminal GFP-tagged constructs were detected in punctuate and vesicular structures distributed throughout the cytoplasm (Figure 7.1.B). A luorescence signal was only weakly detected at the plasma membrane, probably due to the limitations of wide ield microscopy. The punctuate structures of the C-terminal fusion FGFR3-GFP were also observed in bladder cancer cell lines transfected with C-terminal GFP-tagged FGFR3 such as RT112 cells, MGHU3 cells and T24 cells. Furthermore, C-terminal tagged GFP-FGFR3 were able to interact, as non-tagged FGFR3, with HSP90 [START_REF] Laederich | Fibroblast growth factor receptor 3 (fgfr3) is a strong heat shock protein 90 (hsp90) client: implications for therapeutic manipulation[END_REF], suggesting that the GFP tag did not alter the functionality of FGFR3 (data not shown). We therefore used these C-terminal tagged GFP-FGFR3 constructs for following experiments, hereafter called FGFR3-GFP. ). The cells were then lysed and immunoprecipated with anti-FGFR3. In addition, the cell lysate from HeLa expressing WT FGFR3-GFP was incubated with IgG control (IgG). The protein complex was then eluted and analyzed by Western blotting using anti-phosphotyrosine (anti-PY) or anti-FGFR3. Flow through of immunoprecipitation was also loaded to verify the binding eiciency of antibody to antigen.

We irst veriied that HeLa cells expressed WT and mutant FGFR3-GFP at comparable levels (Figure 7.1.A). The functionality of the two GFP fusion proteins was conirmed by evaluating their phosphorylation levels. After 24h transfection with WT or S249C FGFR3-GFP, HeLa cells were treated with DMSO alone (0.1%) or 1 M FGFR in- hibitor PD173074 for 15 min or 1h in medium with 10% serum. Cell lysates were then immunoprecipitated with anti-FGFR3 antibody or control IgG antibodies followed by immunoblotting with anti-FGFR3 antibody to validate the eiciency of immunoprecipitation, and with anti-phosphotyrosine to examine the phosphorylation level of the receptors. The results indicated that both WT and S249C FGFR3 were phosphorylated in medium with 10% serum (Figure 7.1.C). The phosphorylation of FGFR3-GFP was strongly reduced with the treatment of 1 M PD173074 for 15 min and completely inhibited after 1h of treatment. Thus, in HeLa cells transiently expressing FGFR3-GFP, both WT and mutant FGFR3 were activated.

7.2.2

Characterization of the traicking of WT and mutant forms

The possible diference in the cellular distribution of WT and mutant S249C FGFR3 remains poorly explored in the context of cancer. We addressed this question by comparing the localization of these forms in HeLa cells transfected with the FGFR3-GFP constructs described above. We used various antibodies against marker proteins of endosomal compartments, as described in section 6.2.4 (Figure 7.2). In parallel, we cotransfected RAB11A-mcherry with either WT FGFR3-GFP or S249C FGFR3-GFP. The degree of colocalization between FGFR3 and the diferent markers was quantiied by Manders' overlap coeicient (Figure 7.3). The results showed that WT FGFR3-GFP partly colocalized with TFRC, LAMP1 or CD63 (Manders' coeicient= 0.28, 0.30 and 0.6, respectively). There was no signiicant diference between these extent of colocalization (Figures 7.2 and 7.3.A). A small fraction of WT FGFR3-GFP was found in EEA1positive compartment (Manders' coeicient=0.17). Compared to WT FGFR3-GFP, mutant S249C FGFR3-GFP preferentially resided in TFRC-positive compartments (Manders coeicient=0.40) and was less colocalized with LAMP1 or CD63 (Manders' co-eicient=0.2, 0.19 respectively) (Figures 7.2 and 7.3.B). Interestingly, the high colocalization of S249C FGFR3 with TFRC was consistent with the overlap between S249C FGFR3-GFP and RAB11A (Manders' coeicient=0.41) (Figures 7.2 and 7.3.B). Figure 7.3.C shows signiicant diferences in the localization of the two forms of FGFR3 in different compartments. S249C FGFR3 is signiicantly more present in TFRC-or RAB11Apositive compartments than WT FGFR3. Thus, S249C FGFR3 mainly localizes in the endocytic recycling compartment. In contrast, WT FGFR3 can be delivered to the recycling compartment but also to late endosomes and lysosomes. FGFR3 as shown previously. In contrast, in RAB11-depleted cells, we observed a tubulation of TFRC-positive structures (Figure 7.4.A and 7.5.A), as previously reported in other cell lines [START_REF] Ullrich | Rab11 regulates recycling through the pericentriolar recycling endosome[END_REF][START_REF] Haugsten | Photoactivation approaches reveal a role for rab11 in fgfr4 recycling and signalling[END_REF]. In these cells, FGFR3-positive structures appeared larger and mostly concentrated in the perinuclear region than in control cells. In addition, the colocalization with TFRC of the two forms of FGFR3 was signiicantly increased (WT: 66% versus 28% in controls; S249C: 65% versus 39% in controls, Figure 7.6). Of note, the extent of overlap between TFRC with either WT FGFR3 or S249C FGFR3 in RAB11-depleted cells was similar. On the other hand, both forms of FGFR3 were less colocalized with EEA1-positive structures in RAB11-knockdown cells (WT: 9%; S249C: 10%) compared to control cells (WT: 18%; S249C: 18%). Strikingly, the RAB11 depletion induced the accumulation of LAMP1-or CD63-positive structures in the perinuclear region (Figure 7.4.A, Figure 7.5.A). However, the extent of colocalization between these structures with WT or S249C was not afected (Figure 7.6). These data suggest that RAB11A and RAB11B play a role in the recycling of both WT and mutant S249C FGFR3.

7.2.4

Signaling of WT and S249C FGFR3

We further examined whether the diference in the traicking of WT and mutated S249C FGFR3 can have an impact on FGFR3 signaling by using HeLa cells and RAB11depleted HeLa cells expressing these two forms of FGFR3. We thus analyzed the ac-

Materials and methods

Cell culture and RNA interference

HeLa cells were grown at 37 ∘ C under 5% CO2 in DMEM high-glucose Glutamax (Gibco, Life Technologies) supplemented with 10% fetal bovine serum (Lonza).

For RNA interference, cells were transfected with either control siRNA or siRNA targeting RAB11A or RAB11B at 10nM each with Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer's instructions, and cultured for 72h for further manipulations. Depletion eiciency was estimated by RT-qPCR and Western blotting. The siRNAs were purchased from Qiagen: Control siRNA (CGUACGCG-GAAUACUUCGATT (sens), UCGAAGUAUUCCGCGUACGTT (antisens)); RAB11A no6 (AAAUGAGUUUAAUCUGGAATT (sens), UUCCAGAUUAAACUCAUUUCG (antisens)); RAB11B no4 (AGUUCAACCUGGAGAGCAATT (sens), UGCUCUCCAGGU-UGAACUCG (antisens)).

Plasmid constructs

The FGFR3-GFP constructs were generated by subcloning the HindIII-BamHI fragment from pcDNA3.1 containing wild type or mutant (S249C) FGFR3 into pEGFP-N3 (Clon-tech). Full length human wt RAB11A lanked with attB1.1 and attB2.1 were synthesized and inserted into pDONR207 (BP cloning reaction, Invitrogen), then transfered into Gateway compatible versions of pmCherry-C1 (Clontech) (LR cloning reaction, Invitrogen). All constructs were sequence-veriied by Sanger sequencing. HeLa cells were transfected with Fugene HD transfection reagent (Promega) according to the manufacturer's instructions.

Antibodies

Antibodies were purchased from Sigma Aldrich: anti-FGFR3 (F0425), anti-alpha-tubulin (T6199); from Cell Signaling Technology: anti-RAB11A (2413); anti-phospho-p44/42 MAPK (Thr202/Tyr204) (9101); anti-phospho-AKT ser473 (4060); anti-phospho-P38 MAPK (Thr180/Tyr182) (4511); anti-rabbit HRP-linked (7074); anti-mouse HRP-linked (7076) from SantaCruz Biotechnology: EEA1 antibodies (sc-6414); from BD Pharmingen: anti-LAMP1 CD107a (553792); from GeneTex: EasyBlot anti Rabbit IgG HRPlinked (GTX221666-01), EasyBlot anti Mouse IgG HRP-linked (GTX221667-01); from Invitrogen: Goat anti-mouse IgG Alexa Fluor 594 (a11032), anti-Transferrin (136800); from Milipore: anti-phospho-tyrosine (05-321); from Jackson ImmunoResearch: Donkey anti-goat IgG Cy3.

Immunoprecipitation

Cells transfected with FGFR3-GFP for 24h treated with FGFR inhibitor PD173074 (Sigma) or DMSO as indicated in igures. Cells were lysed using lysis bufer (25mM Tris pH 7.5, 50mM NaCl and 1% NP40 and freshly supplemented with Protease and phosphatase inhibitor (Roche)). Cell lysates were clariied by centrifugation and the protein concentration was determined with BCA Protein Assay kit (ThermoFischer). 1 mg protein was precleared with 40 l of protein G agarose beads (50% bead slurry) (GEHealthcare) and then incubated overnight with 20 l of protein G agarose beads (50% bead slurry) and either anti-FGFR3 (F4025, Sigma) or normal Rabbit IgG control (A-105-C, R&D systems). Beads were rinsed four times with lysis bufer. The protein complex was eluted from the beads with Laemmli sample bufer (Biorad) and boiled at 95 ∘ C. The immunoprecipitated proteins were separated by SDS-PAGE using 4-15% Tris-glycine precast gel (Biorad) and analyzed by Western blotting.

Western blotting

Protein extracts were obtained using lysis bufer (50mM Tris-HCl (pH 6.8), 2% SDS, 5% glycerol, DTT 2mM, 2.5 mM EDTA, 2.5 mM EGTA) supplemented with Protease and phosphatase inhibitor (Roche)). Cell lysates were clariied by centrifugation. Protein concentration was determined with a BCA Protein Assay-Reducing Agent Compatible kit (ThermoFischer). Proteins (10 g) were separated by SDS-PAGE using 4-15% Tris-glycine precast gels (Biorad) and transferred to nitrocellulose membranes using Trans-Blot Turbo transfer system (Biorad). Membranes were then blocked with 5% non-fat milk or 5% BSA at room temperature. Membranes were then incubated with the primary antibody overnight at 4 ∘ C and secondary antibody for 45 min at room temperature. Signal detection was performed using the Clarity Western ECL substrates (BioRad) followed with exposure on X-ray ilm (Thermoischer) or detection

Materials and methods 103

with ChemiDoc MP imaging systems using Imagelab software (Biorad).

Immunoluorescence staining

Cells were ixed for 15 min with 4% PFA in PBS at room temperature. After permeabilization with 0.5% saponin (Sigma-Aldrich) and 2% BSA (Sigma-Aldrich), cells were incubated with primary and secondary antibodies before being mounted with Mowiol (Biovalley). Fixed samples were imaged with an upright wideield microscope from Leica equipped with 100X CFIPlanAPo VC oil immersion objective (1.4 NA CFI), a piezoelectric motor (Physik Instrumente) and highly sensitive cooled interlined charge-coupled device (CCD) camera (CoolSNAP HQ2, Photometrics). Z-dimension series of images were taken every 0.2 m by Metamorph software (Molecular Devices). After deconvolution (Meine Algorithm [START_REF] Sibarita | Deconvolution microscopy[END_REF]), colocalization between structures was analyzed with ImageJ 1.50 software [START_REF] Schneider | Nih image to imagej: 25 years of image analysis[END_REF]. Manders' overlap coeicients were calculated using the JACOP plugin for ImageJ [START_REF] Bolte | A guided tour into subcellular colocalization analysis in light microscopy[END_REF].

Real-time quantitative PCR

RNA was extracted from cells with RNAeasy kits (Quiagen). One microgram of total RNA was reverse-transcribed with the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). Predesigned assay was used to quantify human TBP gene expression (Applied Biosystems) and custom assays were used for RAB11A and RAB11B. For custom assays, primers and probes were designed with Probe Finder software via the Universal Probe Library Assay Design Center (Roche). RT-qPCR was carried out with the LightCycler 480 Instrument (Roche) in a 20 l reaction mixture containing 20 ng of reverse-transcribed RNA, 1x LightCycler 480 Probe Master, 25 uM each of the forward and reverse primers and 10 uM of the UPL probe (or 1x the predesigned assay probe). All expression assays were run in the same thermal cycling conditions, including an initial step at 95 ∘ C (10 min), followed by 40 cycles at 95 ∘ C (10 s), 60C (30 s) and 72 ∘ C (10 s). For each gene, the amount of mRNA was normalized to that of the TBP gene by the 2 -∆∆ method. Primers and probes were used for RAB11A: CTGCAC-CTTTGGCTTGTTTT (forward), CAGGGCAGTTCCTACAGATGA (reverse), probe 31; for RAB11B: CGACGAGTACGACTACCTATTCAA (foward), AACTCGTTGCGGGT-GAAG (reverse), probe 62.

Identiication of FGFR3-interaction partners in bladder cancer

8

In order to better elucidate the molecular mechanisms underlying FGFR3 signaling in bladder cancer, we aimed to identify interaction partners of FGFR3. We generated human bladder cancer RT112 cells transiently expressing GFP-tagged FGFR3 to achieve this goal. Following immunoprecipitation of the FGFR3 protein complexes, interaction partners were identiied using mass spectrometry.

Experimental worklow

To verify the eiciency of transfection, we irst transfected RT112 cells with wild type FGFR3 tagged with GFP at its C-terminus or GFP plasmid (control) by using Fugene HD transfection reagent (Promega). The construct FGFR3-GFP is described in section 6.3. 24h after transfection, the cells were either mounted on coverslips or lysed with lysis bufer (25mM Tris pH 7.5, 50mM NaCl and 0.1% NP40 and freshly supplemented with Protease and phosphatase inhibitor (Roche)). The percentage of RT112 transfected cells was low, ∼ 20%. Western blot indicated that the FGFR3-GFP fusion protein was not cleaved (data not shown).

To prepare the cell lysate for immunoprecipitation, a seed culture of RT112 cells (at 70-80% conluence) was split and plated with fresh media the day before transfection and then grown to 50-60% conluency. The RT112 cells were then transfected with wild type FGFR3-GFP or GFP plasmid as control. 24h after transfection, the cells were lysed with lysis bufer. Protein concentration was determined with a BCA Protein Assay kit (ThermoFischer). For immunoprecipitation of the protein complexes, we used GFP-TRAP (gta-20, chromotek) which is a GFP binding protein coupled to a monovalent matrix (agarose beads). This method has many advantages including high binding ainity, no heavy and light chains of antibodies present in Western blots and mass spectrometry analysis. Because the percentage of RT112 transfected cells was low, 4 mg of the lysates were incubated with GFP-TRAP overnight at 4 ∘ C to enrich the amount of immunoprecipitated protein complexes. After four washes with lysis bufer, the beads were incubated in H2O and transferred to be analyzed at the proteomics platform (Institut Jacques Monod). Briely, the beads were trypsinized to digest the baits and the interacting protein. The unfractionated peptides were desalted prior to their analysis using liquid chromatography online coupled to tandem mass spectrometry (LC-MS/MS) (LTQ-Orbitrap Fusion with nano-LC, from ThermoScientiic). Enriched proteins were identiied using MASCOT (Matrix Science) software using a probabilistic scoring algorithm. To reliably estimate the potential partners of FGFR3, we performed two independent experiments. In order to identify actual interaction partners of FGFR3, we took into account both the abundance ratio between FGFR3-GFP versus GFP and the intensities of the identiied proteins. Using a signiicance B value (p<0.05) (Perseus software, Computational systems biochemistry). Our irst replica allowed the identiication of 88 potential partners for FGFR3 (Figure 8.1.A), while the second replica was not technically successful due to the saturation when the sample were analyzed by mass spectrometry system. Therefore, a manual threshold was set to determine which proteins are probably positive in the list of proteins identiied in the second experiment (Figure 8.1.B) (212 proteins). The proteins which were positive in two replicates were uploaded on Ingenuity Pathway Analysis (IPA) developed by Qiagen, then network analysis was performed. The reference set was the IPA knowledge database, all data source in IPA were included for analysis protein-protein interactions. We also searched in the literature for the function of the identiied proteins.

Results

The intersection of 57 proteins identiied from the two experiments are presented in Figure 8.2. Analysis by IPA helped us to establish a network of 35 proteins containing proteins identiied in the mass spectrometry and additional proteins from the IPA database. The additional proteins have direct or indirect relationship with the proteins identiied by mass spectrometry according to the protein-protein interaction algorithm of the IPA knowledge database. Five networks were proposed by IPA.

The irst network (Figure 8.3) contained 23 proteins identiied in mass spectrometry and 12 additional proteins from IPA database. In this network, we found many well-known partners of FGFR3, such as CDC37 and diferent isoforms of HSP90 [START_REF] Laederich | Fibroblast growth factor receptor 3 (fgfr3) is a strong heat shock protein 90 (hsp90) client: implications for therapeutic manipulation[END_REF], RPS6KA3 (known as RSK2) [START_REF] Kang | Fgfr3 activates rsk2 to mediate hematopoietic transformation through tyrosine phosphorylation of rsk2 and activation of the mek/erk pathway[END_REF], HSPG2 and SDC4 [START_REF] Arikawa-Hirasawa | Perlecan is essential for cartilage and cephalic development[END_REF][START_REF] Elfenbein | Syndecan-4 signaling at a glance[END_REF], and STUB1 [START_REF] Laederich | Fibroblast growth factor receptor 3 (fgfr3) is a strong heat shock protein 90 (hsp90) client: implications for therapeutic manipulation[END_REF]. Proteins of the HSP90 and HSP70 chaperone complexes were also identiied, for instance, TRAP1, DNAJC7, PGK1, LLGL1, TOMM70A, RPAP3, CDK5, BAG6, BAG5 and EZR [START_REF] Taipale | A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways[END_REF]. Of note, CDK5 has been shown to phosphorylate Dynamin and to be essential for synaptic vesicle endocytosis [START_REF] Tan | Cdk5 is essential for synaptic vesicle endocytosis[END_REF]. In addition, the results indicated an interaction between FGFR3 and a group of proteins involved in proteasome degradation: PSMD12, PSMA3, PSMA5, UBC, STUB1 and PSMC1. ERK1/2 was not detected in the list of proteins identiied but we found other substrates of ERK1/2 such as RPS6KA3 and RPS6KA1.

The second network (Figure 8.4) contained less proteins identiied in the mass spectrometry experiment (13 proteins) than the irst one. The IPA database added the proteins which are substrates of FGFR3 downstream signaling such as PI3K, AKT, P38, RAS. ESYT1 and STAT6 are also of interest because members of the same family have been reported to interact with FGFR [START_REF] Tremblay | Extended synaptotagmin interaction with the ibroblast growth factor receptor depends on receptor conformation, not catalytic activity[END_REF][START_REF] Osorio | Growth factor and th2 cytokine signaling pathways converge at stat6 to promote arginase expression in progressive experimental visceral leishmaniasis[END_REF].

There are 9 proteins identiied in our mass spectrometry along with 26 proteins from the IPA constituting the third network (Figure 8.5). This network shows that RAB25 expression is negatively regulated by vimentin [START_REF] Vuoriluoto | Vimentin regulates emt induction by slug and oncogenic h-ras and migration by governing axl expression in breast cancer[END_REF]. RAB25 has been shown to interact with PKIG by another mass spectrometry analysis [START_REF] Ewing | Large-scale mapping of human protein-protein interactions by mass spectrometry[END_REF]. Moreover, AP1S1 is a clathrin adaptor-related protein involving in traicking [START_REF] Takatsu | Identiication and characterization of novel clathrin adaptor-related proteins[END_REF], but its function remains Among the proteins of network 4, there is a group of proteins associated with ubiquitination of receptors such as UBC, ATP6V1A, DCAKD, ATXN10 and TIMM50.

The network 5 points to proteins involved in intracellular transport such as different subunits of COP (Coatomer protein complex), RAB1B, GBF1, ARF, USO1 and LMAN2L. These proteins principally localize to the Golgi complex and the endoplasmic reticulum.

Interestingly, the results showed that there was an interaction between FGFR3 and RAB25 and Dynamin 2 (DNM2). This inding supports our results on the role of RAB25 and DNM2 in the traicking and signaling of FGFR3, as described in Chapter 6. Of note, STAT6 is upregulated in tumors associated with mutated FGFR3, in contrast to CDK2 that is downregulated in these tumors (transcriptomic data from our laboratory). Also, ANXA10 is negatively associated with the Multiple Regional Epigenetic Silencing (MRES) phenotype of bladder cancer. ANXA10 is expressed in tumors which do not present the MRES phenotype and which comprise most of the FGFR3 mutated tumors [START_REF] Vallot | A novel epigenetic phenotype associated with the most aggressive pathway of bladder tumor progression[END_REF][START_REF] Biton | Independent component analysis uncovers the landscape of the bladder tumor transcriptome and reveals insights into luminal and basal subtypes[END_REF]. These data suggest that the interaction of FGFR3 with ANXA10, STAT6 and CDK2 should be tested in priority.

Discussion

The proteins were identiied by mass spectrometry from two experiments with different methods setting the threshold due to diferent eiciency of replicate, therefore another replicate should be performed to validate the results obtained.

Although the results obtained are still preliminary, they suggest new partners of FGFR3 that could help our understanding of its signaling in bladder cancer.

IV.

Discussion

In the aim to understand the molecular mechanisms underlying FGFR3 signaling in bladder cancer with a particular focus on the importance of FGFR3 traicking on its signaling, we investigated the potential role of RAB GTPases. We found that proteins of the RAB11 subfamily (RAB11A, RAB11B and RAB25), in particular RAB25, are overexpressed in tumors carrying mutated and translocated FGFR3 compared to tumors without these alterations. Our results indicate that the depletion of RAB11 and RAB25 and two of their efectors, RAB11FIP2 and MYO5B induces an inhibition of cell viability of bladder cancer cell lines expressing activated FGFR3 (mutated or translocated gene) whereas it had little efect on cell viability of other bladder tumor derived cell lines that do not express activated FGFR3. Other RAB such as RAB7 or RAB21, not involved in the recycling process, had no impact on cell viability of cell expressing altered FGFR3. To gain further insight on how RAB11 and RAB25 regulate cell viability in these cells, we irst examined whether RAB11 and RAB25 depletion can alter the traicking of FGFR3. Our results show that RAB11 and RAB25 play an important role in the recycling of FGFR3 and that the inhibition of recycling favors its degradation. We further investigated their role in FGFR3 activity via the activation of downstream signaling pathways (AKT, P38 and ERK1/2) and the expression of FGFR3 target genes (MYC, DUSP6, TIMP2 and GATA3). Our results would favor a model in which the RAB11-and RAB25-mediated recycling of FGFR3 can sustain its signaling by protecting altered FGFR3 from the degradation pathway and providing a unique platform for FGFR3 signaling from the endocytic recycling compartment.

RAB25 expression

It is thought that RAB25 has an epithelial-restricted expression proile [START_REF] Casanova | Association of rab25 and rab11a with the apical recycling system of polarized madin-darby canine kidney cells[END_REF][START_REF] Cheng | The rab25 small gtpase determines aggressiveness of ovarian and breast cancers[END_REF][START_REF] Goldenring | Expression and properties of rab25 in polarized madin-darby canine kidney cells[END_REF][START_REF] Mitra | Rab25 in cancer: a brief update[END_REF]. Gene expression data from our laboratory conirmed this observation in bladder cancer samples. RAB25 as well as MYO5B are highly expressed in normal urothelium and in most low grade and low stage urothelial tumor samples but are weakly expressed in muscle or chorion samples (data not shown). In contrast, RAB11A, RAB11B and RAB11FIP2 are ubiquitously expressed in diferent cell types (data not shown), which is consistent with indings from other studies [START_REF] Kelly | Rab11 proteins in health and disease[END_REF]. In addition, RAB25 expression is highly correlated to genes associated with diferentiated urothelial markers, for ex-ample, ELF3, VSIG2, ERBB3, GRHL3, CLDN7, GATA3, UPK2; and anti-correlated to epithelial-to-mesenchymal transition markers, such as ZEB1, ZEB2, and FYN (data not shown).

Transcriptome analysis in muscle-invasive bladder tumors has revealed that they are heterogeneous and can be grouped into basal and luminal subtypes like breast cancers [START_REF] Choi | Identiication of distinct basal and luminal subtypes of muscle-invasive bladder cancer with diferent sensitivities to frontline chemotherapy[END_REF]. Like in basal breast cancer, basal bladder tumors contain a claudin-low subtype that is enriched with biomarkers characteristic of epithelial-to-mesenchymal transition, for example ZEB1 and ZEB2. Other molecular features characterizing this subgroup are overexpression of STAT3, EGFR, and CDH3. Luminal muscle-invasive bladder cancers are enriched with activating FGFR3 and ERBB3 mutations and ERBB2 ampliications, and their gene expression proiles are controlled by peroxisome proliferatoractivated receptor (PPAR ). In breast cancers, it was shown that RAB25 is upregulated in luminal ER-positive and PR-positive breast tumors [START_REF] Yin | Increased expression of rab25 in breast cancer correlates with lymphatic metastasis[END_REF][START_REF] Mitra | Rab25 in cancer: a brief update[END_REF]. In contrast, RAB25 is mostly lost in basal-like and triple-negative breast tumors, and displays a tumor suppressor activity in MDA-MB-231, a cell line representative of this group [START_REF] Cheng | Loss of rab25 expression in breast cancer[END_REF]. We expected to obtain the same result in bladder tumors. Following RAB25 depletion, the cell viability of UMUC5 cells, a basal-like bladder cancer cell line, was not inhibited, and unexpectedly the same result was obtained in UMUC9 cells, a luminal bladder cancer cell line that depends on PPAR for growth. Our results show that RAB25 along with the other members of the RAB11 subfamily play a protumorigenic role speciically in cell lines presenting FGFR3 alterations. Below, I discuss about the putative role of these RAB GTPases in bladder tumorigenesis.

RAB11/RAB25 GTPases sustain the cell viability via FGFR3 activity

No additive efect on cell viability was observed following simultaneous depletion of RAB11/RAB25 and FGFR3 compared to RAB11/RAB25 or FGFR3 depletion alone, suggesting that RAB11 and RAB25 sustain cell viability via FGFR3 activity. Furthermore, following RAB11/RAB25 knockdown, FGFR3-GFP accumulated in TFRC-positive recycling compartment but the extent of colocalization with LAMP1-or CD63-positive structures was also increased, suggesting that FGFR3 might be degraded. It is possible that the level of total FGFR3 could be partly reduced but not completely depleted in cells knockdown for RAB11/RAB25 compared to control cells. Additionally, compared to control cells, about only 50% of MYC expression was inhibited in diferent conditions of RAB11/RAB25 depletion but about 80% MYC expression was reduced following FGFR3 knockdown. It was shown in our laboratory that in RT112 cells, FGFR3 induces MYC upregulation via stabilizing MYC mRNA and via protecting MYC protein degradation from proteasome [START_REF] Mahe | Caractérisation des voies de signalisation des oncogènes FGFR3 muté et FGFR3-TACC3 dans les carcinomes de vessie[END_REF]. Taken together, in RAB11/RAB25depleted cells, the inhibitory efect on cell viability could relect the efect on MYC expression resulting from a decreased FGFR3 activity. It will be important to estimate the amount of FGFR3 present at the plasma membrane following RAB11/RAB25 depletion by using cell surface biotinylation assays. It will be also necessary to examine the MYC protein level after RAB11/RAB25 silencing.

Proposed working model

From our indings and data from other studies, we propose a model for FGFR3 recycling and signaling in bladder cancer carrying altered FGFR3, illustrated in Figure 9.1.

FGFR3 activates its signaling pathways at the plasma membrane

Our results from the Dyngo4a treatment suggest that AKT and ERK1/2 are activated by FGFR3 from the plasma membrane. This inding is consistent with another study showing that clathrin-dependent endocytosis is not required for eicient activation of FGFR3 on phosphorylation of FRS2, an upstream substrate of AKT in FGFR signaling, and on phosphorylation of ERK1/2 [START_REF] Haugsten | Clathrin-and dynamin-independent endocytosis of fgfr3implications for signalling[END_REF]. As in RT112 cells, FGFR3 is constitutively activated in a ligand stimulation-independent manner, we cannot exclude that AKT or ERK1/2 would be still phosphorylated from endosomes after Dyngo4a treatment if FGFR3 was internalized via other pathways. Indeed, the block of a given endocytic pathway often leads to the activation of others [START_REF] Doherty | Mechanisms of endocytosis[END_REF]. Moreover, in U2OS cells stably expressing wild type FGFR3, it was shown that FGFR3 is internalized via both clathrin-and dynamin-dependence as well as an unknown clathrin-independent endocytic pathway.

To conclude, we need to verify: i) whether, following Dyngo4a treatment, FGFR3 is completely blocked at the plasma membrane; ii) whether phosphorylation of AKT and ERK1/2 only results from signaling at the plasma membrane; and iii) whether on its way from the endoplasmic reticulum to Golgi and the plasma membrane, FGFR3 can activate its signaling pathways. In addition, to better unravel the role of endocytosis on signaling, several methods to alter receptor internalization should be performed such as depletion of clathrin or AP2.

9.3.2

FGFR3 recycles via distinct domains which are speciic for each member of the RAB11 subfamily

This hypothesis comes from the indings that there is no functional redundancy between the RAB11 subfamily proteins in regulating the viability of RT112 and MGHU3 cells. Another line of evidence supporting this hypothesis is that RAB11/RAB25 silencing has diferential efects on three FGFR3 downstream signaling pathways: P38, AKT and ERK1/2.

RAB25 domain

Our results show that when RAB25 is depleted (alone or with RAB11), elevated level of P-P38 is detected. This suggests that FGFR3 is trapped into a microdomain of the recycling compartment from which FGFR3 can be recycled in a RAB25-dependent manner. Also, P38 can be speciically recruited to this domain and phosphorylated by FGFR3. In turn, P-P38 will stabilize MYC mRNA expression as shown in other studies in our laboratory. In cell simultaneously depleted for RAB11/RAB25 and FGFR3, the P-P38 level was about the same level as control cells, suggesting that P38 was phosphorylated at basal level. This amount of P-P38 may be not enough to stabilize the MYC mRNA level because MYC expression was dramatically reduced (∼90%) in cells simultaneously depleted for RAB11/RAB25 and FGFR3 compared to control cells. We cannot exclude the possibility that FGFR3 can regulate MYC expression via another pathway (termed as X pathway in Figure 9.1). Thus, following RAB25 depletion, the decrease in FGFR3 level can result in a decreased activity of the X pathway, leading to the inhibition of MYC expression though an elevated level of P-P38.

Strikingly, DUSP6 expression was strongly reduced when only RAB25 was depleted (50% compared to control siRNA) or slightly reduced in case of the combination with other siRNAs targeting RAB11A or RAB11B (75% and 90%, respectively). DUSP6 plays a negative role in FGF signaling via a negative feed-back loop at the ERK1/2 level. Indeed, FGF signaling induces phosphorylation of ERK1/2, resulting in binding of Ets2, a transcriptional regulator, to the DUSP6 promoter [START_REF] Ekerot | Negativefeedback regulation of fgf signalling by dusp6/mkp-3 is driven by erk1/2 and mediated by ets factor binding to a conserved site within the dusp6/mkp-3 gene promoter[END_REF]. Conversely, when DUSP6 is expressed, this protein in turn inhibits the phosphorylation of ERK1/2 [START_REF] Smith | Negative feedback predominates over cross-regulation to control erk mapk activity in response to fgf signalling in embryos[END_REF]. In our model, after RAB25 silencing, P-ERK1/2 was not altered, suggesting that another mechanism can promote DUSP6 expression. A recent paper has reported that overexpression of RAB25 induces Hypoxia-inducible factor 1 alpha (HIF-1 ) in an oxygen-independent manner in many cancer cell lines [START_REF] Gomez-Roman | Hypoxia-inducible factor 1 alpha is required for the tumourigenic and aggressive phenotype associated with rab25 expression in ovarian cancer[END_REF]. Furthermore, it was shown that HIF-1 regulates DUSP6 expression through mRNA stabilization [START_REF] Bermudez | The dual-speciicity map kinase phosphatases: critical roles in development and cancer[END_REF]. Therefore, a proposed explanation for our indings is that RAB25 depletion reduces HIF-1 expression, leading to a degradation of DUSP6 mRNA expression. Thus, it will be important to estimate the level of HIF-1 after RAB25 knockdown.

RAB11B domain

An increased phosphorylation of ERK1/2 was speciically obtained when RAB11B was depleted. In this condition, FGFR3 could be trapped into a microdomain from which FGFR3 can be recycled in a RAB11B-dependent manner. This domain is distinct from the RAB25 domain described above because it speciically recruits and activates ERK1/2. As mentioned above, the ERK pathway appears as a major positive regulator of DUSP6 mRNA expression in developing embryos and cancer cell lines [START_REF] Smith | Negative feedback predominates over cross-regulation to control erk mapk activity in response to fgf signalling in embryos[END_REF][START_REF] Ekerot | Negativefeedback regulation of fgf signalling by dusp6/mkp-3 is driven by erk1/2 and mediated by ets factor binding to a conserved site within the dusp6/mkp-3 gene promoter[END_REF][START_REF] Bermudez | The dual-speciicity map kinase phosphatases: critical roles in development and cancer[END_REF]. The unmodiied level of DUSP6 expression obtained after RAB11B depletion but not after FGFR3 silencing supports the role of RAB11B in FGFR3-induced ERK1/2 activation. Although we cannot clearly explain why DUSP6 expression remained at the same level compared to control cells, one possibility is that ERK1/2induced DUSP6 expression has already reached a threshold level. This inding also suggests that ERK1/2 can be activated from the recycling endosomes. Along with the results obtained from the experiment with Dyngo4a treatment, it is possible that following RAB11B depletion, a speciic domain for the recruitment of ERK1/2 by FGFR3 is formed on the recycling compartment. To test this hypothesis, a treatment with ERK1/2 inhibitor in cells depleted for RAB11B should be performed. The ERK pathway also regulates DUSP6 expression at the protein level by phosphorylating DUSP6 on Ser159, Ser174 and Ser197 which is followed by the degradation of the phosphatase by the proteasome [START_REF] Marchetti | Extracellular signal-regulated kinases phosphorylate mitogenactivated protein kinase phosphatase 3/dusp6 at serines 159 and 197, two sites critical for its proteasomal degradation[END_REF]. We expect that the DUSP6 mRNA and protein expression will be reduced compared to control cells or cells depleted for RAB11B.

RAB11A domain

This hypothesis comes from the fact that RAB11A depletion alone did not afect the phosphorylation of ERK1/2 or P38. The observation that MYC expression in cells knockdown for only RAB11A was 70% compared to control can be explained by the reduction of the activity of the X pathway resulting from a decreased in the total level of FGFR3 protein. Strikingly, an increase of DUSP6 mRNA level (140% compared to control cells) was obtained after RAB11A silencing. DUSP6 mRNA stability was shown to be positively regulated by HIF-1 as mentioned above, but also to be negatively regulated by Tristetraprolin (TTP) and Pumilio RNA-binding Family Member 2 (PUM2) [START_REF] Bermudez | The dual-speciicity map kinase phosphatases: critical roles in development and cancer[END_REF]. Whether RAB11A depletion can alter the function of these proteins should be investigated. In the case of triple-RAB11 depletion, a balance between the inhibitory and activating efects on DUSP6 expression is possibly established through diferent pathways.

9.3.3

Evaluation of the proposed model

The fact that RAB proteins localize to distinct endosomal microdomains even on the same compartment has been demonstrated for early and recycling endosomes. These endosomes are formed of multiple RAB4, RAB5 and RAB11 domains that are dynamic but do not signiicantly intermix overtime [START_REF] Sönnichsen | Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of rab4, rab5, and rab11[END_REF]. Three main domains have been observed: one that contains only RAB5, a second containing RAB4 and RAB5, and a third containing RAB4 and RAB11. Similarly, late endosomes contain distinct membrane domains that are positive for either RAB7 or RAB9 [START_REF] Barbero | Visualization of rab9-mediated vesicle transport from endosomes to the trans-golgi in living cells[END_REF]. Recently, it was proposed that RAB11A and RAB11B play distinct roles in the recycling of FGFR4 or PAR-1 [START_REF] Haugsten | Photoactivation approaches reveal a role for rab11 in fgfr4 recycling and signalling[END_REF][START_REF] Grimsey | Recycling and endosomal sorting of protease-activated receptor-1 is distinctly regulated by rab11a and rab11b proteins[END_REF]. Our proposed model can be tested by super resolution luorescence microscopy.

Although we did not directly quantify the protein level of FGFR3 following RAB11 and RAB25 depletion, our results suggest that the FGFR3 level is decreased. However, it is not clear whether RAB11A or RAB11B or RAB25 domains can recruit FGFR3 downstream substrates such as P38 or ERK1/2 under normal condition in RT112 cells or if this recruitment only occurs in cells depleted for RAB11/RAB25. Taken together, our indings clearly indicate that RAB11/RAB25 play an important role in FGFR3 recycling, resulting in sustaining its signaling.

9.3.4

Questions that remain to be elucidated First, the elevated level of P-AKT was observed in cells depleted for only one of the RAB11 subfamily proteins but not in case of double or triple knockdown. Because the Dyngo4a experiment suggests that AKT is phosphorylated at the plasma membrane, we propose that the endocytic recycling network probably creates a microdomain near the plasma membrane where AKT can be recruited following siRNA targeting only one of the RAB11 genes.

Two other FGFR3 target genes, GATA3 and TIMP2, displayed a mRNA expression after RAB11/RAB25 silencing diferent from that obtained after FGFR3 silencing. To our knowledge, the upstream regulation pathway of these genes remains unknown. We thus propose to examine the efects on the expression of GATA3 and TIMP2 after RAB11/RAB25 depletion and a treatment with kinase inhibitors of AKT, P38 and ERK1/2.

9.3.5

Limitations of the study

The quantiication of the expression of FGFR3 target genes was performed in only one RT-qPCR experiment and needs to be repeated. As reverse-transcribed RNAs are already available after diferent siRNA transfection, these experiments can be soon performed. We had to investigate the traicking of FGFR3 in bladder cancer cell lines by using RT112 cells transiently transfected with wild type FGFR3-GFP due to the lack of antibodies speciically recognizing endogenous FGFR3 in immunocytology experiments. Nevertheless, the pattern of subcellular distribution of FGFR3-GFP in RT112 9.4 Traicking and signaling of WT and mutated S249C FGFR3 123 cells is consistent with other studies [START_REF] Haugsten | Diferent intracellular traicking of fgf1 endocytosed by the four homologous fgf receptors[END_REF][START_REF] Degnin | Ligand activation leads to regulated intramembrane proteolysis of ibroblast growth factor receptor 3[END_REF]. The GFP fusion protein seems to be functional in these cells since it interacts with HSP90, a well-known chaperone partner of FGFR3 [START_REF] Laederich | Fibroblast growth factor receptor 3 (fgfr3) is a strong heat shock protein 90 (hsp90) client: implications for therapeutic manipulation[END_REF]. Data from our laboratory indicate that in RT112 cells, wild type FGFR3 forms heterodimers with the fusion protein FGFR3-TACC3, suggesting that wild type FGFR-GFP introduced in these cells can interact together with endogenous wild type or translocated FGFR3. We did not choose the labeled ligand stimulation method to follow the traicking of FGFR3 due to the fact that in RT112 cells displaying altered FGFR3, FGFR3 is constitutively activated and no increase in phosphorylation could be achieved in presence of FGF1 ligand, suggesting that the receptor internalization is independent of ligand stimulation. Also, there is no ligand speciic for the FGFR3b isoform which was expressed in bladder cancer cell lines and other FGFRs have the same ainity binding with FGF1. As mentioned before, it will be important to quantify the FGFR3 protein level and the proportion of FGFR3 recycled to conirm the proposed hypothesis.

9.4

Traicking and signaling of WT and mutated S249C FGFR3 9.4.1

Diference between the subcellular distribution of WT and mutated S249C FGFR3

In an attempt to characterize the potential diference in the traicking of wild type and mutant FGFR3, we quantiied the extent of colocalization between FGFR3 and diferent markers of intracellular compartments in HeLa cells transiently expressing FGFR3-GFP (WT or S249C forms). As HeLa cells do not express detectable levels of FGFR3, FGFR3-GFP cannot interact with endogenous protein. FGFR3-GFP displayed a vesicular pattern dispersed throughout the cytoplasm. We also observed this vesicular structure in several bladder cancer cell lines (RT112, MGHU3, T24, HT1376). This subcellular distribution is consistent with several studies, for example that of WT and K644E/K650M FGFR3 in Cos-7 stably expressing FGFR3-GFP [START_REF] Cho | Defective lysosomal targeting of activated ibroblast growth factor receptor 3 in achondroplasia[END_REF][START_REF] Degnin | Ligand activation leads to regulated intramembrane proteolysis of ibroblast growth factor receptor 3[END_REF]. Cy3-labeled FGF1 is internalized in vesicular structures in U2OS cells stably expressing FGFR3, suggesting that FGFR3 is found in endosomal compartments [START_REF] Haugsten | Clathrin-and dynamin-independent endocytosis of fgfr3implications for signalling[END_REF]. However, in our study, we could not compare the subcellular localization of FGFR3-GFP with that of endogenous FGFR3 in bladder cancer cells due to the lack of anti-FGFR3 antibodies speciically recognizing endogenous FGFR3 by immunoluorescence staining.

Our results indicate that the subcellular distribution of WT FGFR3 difers from that of mutated S249C FGFR3. At steady state, activated WT FGFR3 localizes in several compartments including early endosomes, late endosomes/lysosomes and also the recycling compartment. There was no signiicant diference between the overlap of FGFR3 with late endosomes and the recycling compartment. In contrast, activated S249C resides preferentially in the recycling compartment, and only a small fraction of mutated FGFR3 was found in late endosomes/lysosomes. This pattern of distribution of WT and mutant FGFR3 was also reported in other studies. Indeed, in Cos-7 stably expressing WT or mutant FGFR3, activated WT FGFR3 is normally targeted for lysosomal degradation through a mechanism involving c-Cbl-mediated ubiquitination [START_REF] Cho | Defective lysosomal targeting of activated ibroblast growth factor receptor 3 in achondroplasia[END_REF]; in contrast, mutant forms (K644E, K650M) of FGFR3 escape from lysosomal compart-ment to enter into a recycling pathway. The consequence of this derailed traicking is the ampliication of the normal inhibitory FGFR3 signals in the skeletal growth plate. The authors suggest that a defect in c-Cbl-mediated ubiquitination of mutant FGFR3 is the cause of this altered traicking. However, the question of the ubiquitination of mutant FGFR3 is still under debate [START_REF] Cho | Defective lysosomal targeting of activated ibroblast growth factor receptor 3 in achondroplasia[END_REF][START_REF] Bonaventure | The localization of fgfr3 mutations causing thanatophoric dysplasia type i diferentially afects phosphorylation, processing and ubiquitylation of the receptor[END_REF][START_REF] Monsonego-Ornan | The transmembrane mutation g380r in ibroblast growth factor receptor 3 uncouples ligand-mediated receptor activation from down-regulation[END_REF]. Also, the TDI mutant (R248C) localizes in a perinuclear region and colocalizes with nucleoporin; by contrast only a partial or no overlap with late endosomes/lysosomes was observed [START_REF] Legeai-Mallet | Fibroblast growth factor receptor 3 mutations promote apoptosis but do not alter chondrocyte proliferation in thanatophoric dysplasia[END_REF].

However, our indings on the subcellular distribution of FGFR3 difer from those of Bonaventure et al [START_REF] Bonaventure | The localization of fgfr3 mutations causing thanatophoric dysplasia type i diferentially afects phosphorylation, processing and ubiquitylation of the receptor[END_REF]. Wild type FGFR3 was shown to colocalize with GM130, a cis-Golgi marker, in 293-VnR cells [START_REF] Bonaventure | The localization of fgfr3 mutations causing thanatophoric dysplasia type i diferentially afects phosphorylation, processing and ubiquitylation of the receptor[END_REF]. Mutated FGFR3 (R248C, Y373C) showed partial colocalization with peptidyl disulide isomerase (PD1), an endoplasmic reticulum marker. These diferences may be due to the two diferent cell types used (293-VnR cells, a human embryonic kidney cells expressing the vitronectin receptor and HeLa cells, a human cervix carcinoma cell line). Our results also difer from those of Lievens et al [START_REF] Lievens | The thanatophoric dysplasia type ii mutation hampers complete maturation of ibroblast growth factor receptor 3 (fgfr3), which activates signal transducer and activator of transcription 1 (stat1) from the endoplasmic reticulum[END_REF] that reported that WT FGFR3 localizes mainly at the plasma membrane in HEK293 cells transiently expressing FGFR3-GFP. This diference may be due to the fact that in our model WT FGFR3-GFP is always phosphorylated and activated; in contrast, in their model, WT FGFR3-GFP is not phosphorylated, suggesting that the receptor cannot be internalized.

Aside from S249C FGFR3, we wanted to compare the traicking of FGFR3-TACC3, a fusion FGFR3 protein recently identiied in bladder cancer but also many other cancer type such as glioblastoma and lung cancer [START_REF] Singh | Transforming fusions of fgfr and tacc genes in human glioblastoma[END_REF][START_REF] Wu | Identiication of targetable fgfr gene fusions in diverse cancers[END_REF]. Unfortunately, the PCR ampliication of this fusion from RT112 cells failed, maybe due to the length of this gene. Several kits with diferent types of Taq Polymerase will be tested to obtain this construct.

9.4.2

Signaling of WT and S249C FGFR3

In our studies, we could not conclude whether there was a diference between WT and S249C FGFR3 because the introduction of FGFR3 in HeLa cells did not induce a modiication in the AKT, P38 and ERK1/2 downstream signaling pathways of FGFR3. Also, no diference in the phosphorylation of these proteins was observed in cells expressing wild type and S249C FGFR3-GFP. We propose to use human bladder cancer cell lines to directly investigate the impact of the diferent forms of FGFR3 on traficking and signaling. These cells will provide the machinery required for FGFR3 transforming activity. For example, MGHU3 cells are dependent on constitutively activated FGFR3 for growth and transformation and expressed weakly mutated FGFR3. However MGHU3 cells are diicult to transfect by using cationic lipid reagents. Thus, electroporation or lentivirus could be better transfection strategy for this cell line.

In conclusion, our results indicate that RAB25 and RAB11 GTPases play an important role in the recycling of FGFR3. They help sustain cell viability by regulating the amplitude of FGFR3 response and probably provide a speciic platform for its activity. Our study contributes to understand the molecular mechanisms underlying the relationships between the traicking and signaling of FGFR3 in the context of bladder cancer. It could pave the way for the identiication of new targets and new therapeutic

Rôle des GTPases RAB25 et RAB11 dans la tumorigénèse des cancers de la vessie

Résumé : L'activation constitutive de FGFR3 par mutation ou translocation est l'un des évènements les plus fréquents dans le cancer de la vessie. Une dérégulation de RAB25, une protéine impliquée dans le processus de recyclage des récepteurs de surface, a été montrée dans diférents cancers. Des données du transcriptome des cancers de vessie ont montré que RAB25 est surexprimé dans les tumeurs présentant des altérations de FGFR3. L'objectif de cette thèse a été d'étudier l'implication possible de RAB25, des protéines de la même famille, RAB11A et RAB11B, et leur efecteurs RAB11FIP2 et MYO5B dans 1) la tumorigénèse des tumeurs altérées pour FGFR3 et 2) le traic et la signalisation de FGFR3. Nos résultats montrent que l'extinction de ces protéines par des siARNs induit une diminution signiicative de la viabilité cellulaire des cellules exprimant des formes constitutivement activées de FGFR3. Les efets de la déplétion de RAB25 et RAB11 sur le recyclage de FGFR3, sur les voies de signalisation de FGFR3 et sur l'expression des gènes cibles de FGFR3 suggèrent que le recyclage de FGFR3 régulé par RAB25 et RAB11 peut prolonger le signal de FGFR3 et peut fournir une plateforme pour la signalisation de FGFR3. Nous avons également comparé la distribution cellulaire des formes sauvage et muté (S249C) de FGFR3 portant une étiquette GFP dans des cellules HeLa. Les deux formes de FGFR3 se trouvent dans plusieurs compartiments intracellulaires mais FGFR3 muté se localise préférentiellement dans le compartiment de recyclage. Ce projet nous a permis de mieux caractériser la traic de FGFR3 dans le cancer de la vessie et son lien avec la signalisation et l'activité de FGFR3.

Mots-clés : FGFR3, cancer de la vessie, recyclage, GTPases RAB, sous-famille RAB11

Role of RAB25 and RAB11 GTPases in bladder tumorigenesis

Abstract : Activation of FGFR3 by point mutation, translocation and overexpression is one of the most frequent events in bladder cancer. The dysfunction of RAB25, a GTPase involved in endocytic recycling of transmembrane receptor, has been shown in many cancers. Gene expression data in bladder cancer indicates that RAB25 expression is significant higher in tumors carrying altered FGFR3. The thesis project aimed to investigate the potential role of RAB25, proteins from the same family (RAB11A and RAB11B) and their efectors RAB11FIP2 and MYO5B in 1) the tumorigenesis of tumors carrying altered FGFR3 and 2) the traicking and the signaling of FGFR3. Our results demonstrate that depletion of these proteins by siRNA signiicantly reduces cell viability in cells expressing constitutively activated forms of FGFR3. The efects of RAB25 and RAB11 silencing on FGFR3 traicking and signaling and the expression of FGFR3 target genes suggest that the RAB11-and RAB25-mediated recycling can sustain the signaling by protecting altered FGFR3 from the degradation pathway, and can provide a platform for FGFR3 signaling We also compared the subcellular distribution of wild type and mutant (S249C) forms of FGFR3. These two forms localize to diferent compartments including early endosomes, late endosomes and recycling compartments. The S249C FGFR3 mutant preferentially localizes to the endocytic recycling compartment. Our indings shed light to the molecular mechanisms underlying the relationships between the traicking and signaling of FGFR3 in the context of bladder cancer. Keywords : FGFR3, bladder cancer, oncogene, recycling, RAB GTPases, RAB11 subfamily

7. 1

 1 Generation of wild type and S249C FGFR3-GFP expressing HeLa cells 94 7.2 Subcellular localization of WT and S249C FGFR3 96 7.3 Subcellular localization of WT and S249C FGFR3, Manders' overlap coeicients 97 7.4 WT FGFR3 accumulates in TFRC-positive structures in RAB11-depleted cells 98 7.5 S249C FGFR3 accumulates in TFRC-positive structures in RAB11-depleted cells 99 7.6 FGFR3 accumulates in TFRC-positive structures in RAB11-depleted cells, Manders' overlap coeicients 100 7.7 Signaling of WT and S249C FGFR in HeLa cells 101 8.1 Scatter plot of the protein abundance ratios against the intensities of the identiied proteins 106 8.2 List of proteins identiied in mass spectrometry 107 8.3 Network 1 109 8.4 Network 2 110 I.

Figure 1 . 1

 11 Figure 1.1 Layers of the bladder wall. Left. Cross-section of the bladder wall stained with hematoxylin/eosin (objective of 10X). The bladder wall showing a number of folds is composed of several distinct layers: adventitia, muscularis propria, lamina propria, urothelium (in contact with lumen). Right. Higher magniication (objective of 40X) of yellow frame on the left. Urothelium consist of diferent cell types, outside to inside: umbrella, intermediate and basal cells [2].

Figure 1 .

 1 Figure 1.2 TNM classiication of carcinomas of the urinary bladder[START_REF] Eble | Who classiication of ttumour. pathology and genetic of tumour of the urinary system and male genital organs[END_REF].

Figure 1 . 3

 13 Figure 1.3 Bladder cancer grading and staging. a. Staging of bladder cancer according to TNM system is shown. b. Grading according to 1973 WHO and 2004 WHO/ISUP criteria is shown. The major diference is in the classiication of papillary tumors, which are classiied as grades 1, 2 and 3 in the older system and as papillary urothelial malignancy of low malignant potential (PUNLMP; equivalent to grade 1), low-grade or high-grade papillary urothelial carcinoma in the WHO/ISUP 2004 classiication [104].

Figure 1 . 4

 14 Figure 1.4 Potential pathogenesis pathways are shown based on histopathological and molecular observations. The blue and purple pathways indicate the two major pathways with distinct histopathological and molecular features that have been recognized for the past two decades. Percentages at diagnosis and key molecular features are indicated. The recent identiication of multiple distinct molecular subtypes of NMIBC and MIBC suggests multiple subpathways within each of the major pathways.It should be noted that the existence of multiple subtypes within all of the histologically recognizable intermediates remains hypothetical. Development of histopathologically recognizable urothelial alterations is preceded by clonal expansion of altered cells within the urothelium. Low-grade papillary tumours may arise via simple hyperplasia and minimal dysplasia, and these are characterized at the molecular level by loss of heterozygosity (LOH) of chromosome 9 and activating mutations of ibroblast growth factor receptor 3 (FGFR3), PIK3CA (which encodes p110 of PI3K) and stromal antigen 2 (STAG2). These non-invasive tumours recur frequently but are genetically relatively stable. Muscle -invasive carcinoma is thought to arise mainly via lat dysplasia and carcinoma in situ (CIS), which commonly show TP53 mutation in addition to chromosome 9 deletions but no FGFR3 mutations. Muscle-invasive tumours are genetically unstable and accumulate many genomic alterations. Uncertainty about the possible development of high-grade noninvasive papillary tumours from lat dysplasia is indicated by a dashed arrow. The inding of a subtype of invasive carcinomas with FGFR3 mutation and loss of cyclin-dependent kinase inhibitor 2A (CDKN2A) may suggest a route by which low-grade non-invasive papillary tumours can progress to muscle invasion (dashed arrow). ARID1A, AT-rich interactive domain 1A; EMT, epithelial-mesenchymal transition; RHOGDI1, RHO-GDP dissociation inhibitor 2; ZEB1, zinc-inger E-box binding homeobox 1[START_REF] Knowles | Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity[END_REF].

Figure 1 . 5

 15 Figure1.5 PI3K/AKT/mTOR pathway. mTORC1 is activated by growth factors through the PI3K/AKT signaling pathway which phosphorylates and inhibits TSC1/TSC2 complex. This complex is an upstream negative regulator and acts as a GTPase-activating protein for Rheb GTPase. The activation of Rheb consequently promotes the mTOR activity which targets several downstream proteins such as 4E-BP and S6K. 4E-BP is a family of translational repressor proteins which interacts with eukaryotic translation initiation factor (eIF4E) depending their phosphorylation level. When hyperphosphorylated, 4e-BP can not bind to eIF4E which results in the recruitment of the translation initiation factor. S6K, for Ribosomal Protein S6 Kinase, regulates mRNA translation by activating or inhibiting some substrates, e.g. Ribosomal protein S6, eIF4B, eEF2K. These activities of mTOR on translational control result in the positive regulate cell growth and proliferation[START_REF] Costa-Mattioli | mtor complexes in neurodevelopmental and neuropsychiatric disorders[END_REF].

Figure 2 . 2 FibroblastFigure 2 . 3 FGFR

 2223 Figure 2.2 Receptor Tyrosine Kinase families. Human RTKs contain 20 subfamilies, shown here schematically with the family members listed beneath each receptor. Structural domains in the extracellular regions, identiied by structure determination or sequence analysis, are marked according to the key. The intracellular domains are shown as red rectangles [117].

Figure 2 . 4 FGF

 24 Figure 2.4 FGF signaling pathways. Binding of paracrine FGFs to FGFR with heparan sulfate (HS)and/or Heparan Sulfate proteoglycans (HSPG) as cofactor induces the formation of ternary FGF-heparin-FGFR complex, which activated the FGFR intracellular TK domain by phosphorylation of speciic tyrosine residues. The activated receptor is coupled to intracellular signaling pathways including the RAS-MAPK, PI3K-AKT, PLC , and STAT pathways[START_REF] Ornitz | The ibroblast growth factor signaling pathway[END_REF].

Figure 2 . 5

 25 Figure 2.5 Phospholipase C pathway. Activated PLC cleaves PIP2 to produce IP3 and DAG [1].

Figure 2 . 6

 26 Figure 2.6 FGF and FGFR gene expression in developing endochondral bone. (A) FGFR gene expression.FGFR1 is expressed in limb mesenchyme (M) and in the periphery of the mesenchymal condensation (pink). FGFR2 is irst expressed in condensing mesenchyme (purple). FGFR3 expression is initiated as chondrocytes diferentiate and proliferate (green). FGFR1 expression is present as chondrocytes hypertrophy (red), and FGFR2 expression is prominent in osteoblasts in the ossiication center (purple). Both FGFR1 and FGFR2 appear to be coexpressed in the perichondrium (pink). (B) FGF gene expression. At the limb bud stage, FGF10 is expressed in distal mesenchyme (M) (yellow) and FGF8 is expressed in the overlying apical ectodermal ridge (AER) (pink). FGF2 and FGF9 expression is observed in the developing condensation (blue), and FGF18 expression is observed in the perichondrium and presumptive joint positions (green). FGF7 is expressed in loose mesenchyme and perichondrium surrounding the mesenchymal condensation (tan)[START_REF] Ornitz | Fgf signaling in the developing endochondral skeleton[END_REF].

Figure 2 . 7

 27 Figure 2.7 Signaling pathways activated by FGF/FGFR2 control osteoblast proliferation and diferentiation [129].

Figure 2 . 8 FGFR3

 28 Figure 2.8 FGFR3 mutations associated with skeletal disorders. The locations of the missense mutations associated with skeletal dysplasias: Hypochondroplasia (HCH), achondroplasia (ACH), severe ACH with developmental delay and acanthosis nigricans (SADDAN), thanatophoric dysplasia (TDI and TDII), Crouzono syndrome zith acanthosis nigricans (C+AN), Muenke craniosynostosis (MC) [217].

Figure 2 . 9

 29 Figure 2.9 Flow chart for the classiication of the suggested crucial factors concerning the pathogenesis or severity of the FGFR3-related skeletal dysplasias. Continuous phosphorylation either dependent or independent on ligand stimulation is the crucial factor to distinct mutated FGFR3s from WT-FGFR3. Among mutants, ER localization of the receptor and phosphorylation level of mutated FGFR3 are the factors which determine the severity of the skeletal dysplasias [78].

Figure 2 .

 2 Figure 2.10 Molecular mechanisms of FGFR3 signaling in cartilage. Aberrant activation of FGFR3alters chondrocyte behavior by inducing premature growth arrest, loss of extracellular matrix, altered differentiation, and changes in cell shape. Together, these cellular phenotypes (gray arrows) contribute to profound disruption of the growth plate cartilage resulting in skeletal dysplasia. At the molecular level, the growth arrest phenotype is mediated by induction of several inhibitors of the cell cycle, belonging to cip/kip family (p21) or INK4 family (p16, p18, p19), whereas the loss of the extracellular matrix originates from both inhibition of production of major matrix components (collagen type II and aggrecan), and active matrix degradation, mediated by several members of MMP family. Expression of two important physiological regulators of chondrocyte diferentiation, Indian hedgehog (Ihh) and parathyroid hormone-related protein (PTHrP), is inhibited by FGFR3 in cartilage, likely contributing to impaired chondrocyte hypertrophic diferentiation. ERK MAP kinase is a major pathway for growth arrest, extracellular matrix loss, and impaired chondrocyte diferentiation. FGFR3 causes prolonged activation of the ERK signaling module (RAS-RAF-MEK-ERK), mediated by adapter (GAB1, FRS2, and SHC) driven recruitment of SHP2-GRB2-SOS1 complexes to the cell membrane, where they activate RAS. In addition, SNAIL1 transcription factor is involved in regulation of FGFR3-mediated ERK activity, although the exact nature of this regulation is not presently clear (question marks). The FGFR3-mediated activation of the ERK pathway is antagonized by CNP signaling, which inhibits ERK pathway by inactivation of RAF kinase, via inhibitory phosphorylation mediated by cGMP-activated protein kinase (PKG). Some FGFR3 mutants also activate STAT1, possibly via direct phosphorylation at Y701. It is, however, currently unclear whether activated STAT1 or other STATs induce cell cycle inhibitor expression in cartilage, thereby contributing to the FGFR3-mediated growth arrest (question mark). Finally, chronic activation of FGFR3 leads to inhibition of canonical cytokine-STAT signaling in chondrocytes, via both induction of SOCS inhibitors of cytokine signaling and inhibition of expression of receptors for IL6 (IL6R ) or LIF (LIFR). As the latter cytokines represent positive regulators of chondrocyte proliferation, their inhibition might contribute to the pathological efects of FGFR3. NPR-B, natriuretic peptide receptor B/guanylyl cyclase B; GTP, guanosine-5'-triphosphate; cGMP, cyclic guanosine monophosphate; gp130, glycoprotein 130[START_REF] Foldynova-Trantirkova | Sixteen years and counting: the current understanding of ibroblast growth factor receptor 3 (fgfr3) signaling in skeletal dysplasias[END_REF].

Figure 2 . 12

 212 Figure 2.12 Relative frequencies and positions of FGFR3 mutations in bladder cancers [65].

Figure 2 .

 2 Figure 2.13 Schematic representation of two FGFR3 fusions identiied in bladder cancers. The most common breakpoint of each fusion is shown. Occurence numbers (n) indicate the total number of times the fusion has been identiied, including breakpoints not shown in the igure [60].

Figure 3 . 1

 31 Figure 3.1 Morphological and molecular summary of the endocytic pathways [47].

3. 1 39 Figure 3 . 2

 13932 Figure 3.2 The proposed ive steps of clathrin-coated vesicle formation. Nucleation: FCHO proteins bind phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2)-rich zones of the plasma membrane and recruit EPS15-EPS15R and Intersectins to initiate clathrin-coated pit formation by recruiting AP2. Cargo selection:AP2 recruits several classes of receptors directly through its -subunit and -subunit. Cargo-speciic adaptors (for example, stonin, HRB and Numb) bind to AP2 appendage domains and recruit speciic receptors to the AP2 hub. Coat assembly: clathrin triskelia are recruited by the AP2 hub and polymerize in hexagons and pentagons to form the clathrin coat around the nascent pit. Scission: the GTPase dynamin is recruited at the neck of the forming vesicle by BAR domain-containing proteins, where it self-polymerizes and, upon GTP hydrolysis, induces membrane scission. The actin machinery module can be added at this stage for actin polymerization at the neck of the pit, which can aid in vesicle production (not shown). Uncoating: auxilin or cyclin G-associated kinase (GAK) recruit HSC70 to disassemble the clathrin coat and produce an endocytic vesicle containing the cargo molecules. Synaptojanin probably facilitates this by releasing adaptor proteins from the vesicle membrane through its PtdIns lipid phosphatase activity. The components of the clathrin machinery are then freed and become available for another round of CCV formation[START_REF] Mcmahon | Molecular mechanism and physiological functions of clathrin-mediated endocytosis[END_REF].

Figure 3 . 3

 33 Figure 3.3 Caveolae composition and their links to stress ibers and the actin cytoskeleton.The main molecules that shape caveolae (caveolins, cavins and pacsin2) and the proteins that regulate their dynamics (Dyn2, EHD2 and ilamin A) are depicted. The caveolar molecules that have functional or physical association with the actin cytoskeleton, and therefore potentially mediate the physical and functional interaction between caveolae and actin ibers, are also indicated. Filamin A is depicted here as the main protein mediating a linkage with stress ibers, but other yet unidentiied linkers might exist (indicated as unknown linker). Regulators of stress ibers (Abl kinases and mDia1) that impinge on caveolae organization and traicking are shown next to RhoA, the main regulator of stress ibers, which is regulated by Cav1[START_REF] Echarri | Caveolae -mechanosensitive membrane invaginations linked to actin ilaments[END_REF].

Figure 3 . 4

 34 Figure 3.4 Post-endocytic traicking of signaling receptors. See further detail in the text [181].

Figure 3 . 6

 36 Figure 3.6 Pathways of endocytosis and endocytic recycling. Itinerary of cargo proteins entering cells by clathrin-dependent (blue cargo) and clathrin-independent (red cargo) endocytosis. Clathrin-dependent cargoes can recycle back to the cell surface through a rapid recycling pathway that requires RAB4 and RAB35. Both types of cargo can move from the early endosome to the ERC by a process that requires sorting nexin 4 (SNX4), dynein, RAB10, RAB22A, RAB11FIP2 (in complex with carboxy-terminal epidermal growth factor receptor substrate 15 homology domain-containing protein 3 (EHD3)), RAB11FIP3 (in complex with ARF6 or RAB11) and RAB11FIP5. From the ERC, recycling of both types of cargo requires RAB11, and recycling of clathrin-independent cargoes involves the generation of distinctive RAB8-and RAB22Adependent tubules, in addition to many other factors. Some clathrin-dependent cargoes might also recycle through these diferent pathways. RAB10, RAB11, RAB22A and RAB35 are associated with the tubular recycling endosome and, along with EHD1 (homologous to receptor mediated endocytosis protein 1 (RME-1) in C. elegans) in complex with the mammalian apoptosis-linked gene 2-interacting protein X1 homologue ALIX (also known as PDCD6IP) and RAB11FIP2, are required for recycling. In the periphery, the tubules seem to break up into vesicles prior to fusing with the cell surface, a process that initially requires partitioning defective protein 3 (PAR3) and CDC42 and subsequently ADP-ribosylation factor 6 (ARF6), RAB11 and cortical actin. This is a composite description of endocytic recycling and all of the components shown here may not be evident in a given cell type. Adapted from[START_REF] Grant | Pathways and mechanisms of endocytic recycling[END_REF].

Figure 3 . 7

 37 Figure 3.7 Signaling processes begin at the cell surface and continue in endosomes. a Activation of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)-Akt signaling cascades byreceptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) occurs at the plasma membrane and in early, recycling and late endosomes. In all of these locations activated, phosphorylated MAPK dissociates from the MAPK kinase (MK), unless stably associated with a scafold protein such as betaarrestin, and phosphorylates substrates in the cytoplasm and nucleus. When GPCRs are stimulated by lysophosphatidic acid, the activated G protein subcomplex Gbeta-Ggamma can activate PI3K and Akt at the plasma membrane and also translocate to the early and recycling endosomes to activate endosomal PI3K gamma-isoform, which leads to the phosphorylation and activation of Akt in endosomes. b Model of retrograde endosome signaling in neurons. Ligand-bound nerve growth factor receptors (NTRKs) are internalized by clathrin-dependent and clathrin-independent endocytosis, with associated components of the MAPK signaling cascade, into early endosomes or macropinosomes that have a multivesicular body-like morphology. NTRK signaling complexes are delivered to the soma in retrograde endosomal carriers, which are a population of early or late endosomes and macropinosomes, by dynein motor-mediated microtubular transport. The MAPK extracellular signal-regulated kinase 5 (ERK5) is then phosphorylated and activated in the soma. It phosphorylates the cAMP responsive element-binding protein (CREB), which regulates the transcription of anti-apoptotic genes. Grey arrows show traicking pathways. Black arrows show protein translocations or complex assembly. GRB2, growth factor receptor-bound protein 2; MP1, MEK1 partner 1; PtdIns(3,4,5)P3, phosphatidylinositol-3,4,5-trisphosphate; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; SH2, Src homology 2.[START_REF] Sorkin | Endocytosis and signalling: intertwining molecular networks[END_REF].

Figure 4 . 1

 41 Figure 4.1 Structural features of RAB GTPases. (a) Ribbon drawing of RAB3A complexed with the GTP analog GppNHp. Purple, bound nucleotide; orange sphere, Mg2+ ion; blue, switch I and II regions; green, helices and sheets; yellow, loops. (b)A proile amino-acid sequence of the RAB GTPase subfamily generated using the hidden Markov model (HMM) method. The uppercase/lowercase coding represents outcome of the proile HMM method. Upper-case characters, residues found in the proile with a probability of p<0.5; red, RAB-speciic residues (RABF1-5); dark blue, subfamily-speciic motifs (RABSF1-4); cyan, highly conserved nucleotide-binding motifs; G, guanine-base-binding motif; PM, phosphate/magnesiumbinding motif. The secondary structure units ( helices, strands, and loops, ) are indicated above the sequence[START_REF] Stenmark | The rab gtpase family[END_REF].

Figure 4 .

 4 Figure 4.2 The RAB cycle.The newly synthesized RAB protein associates with RAB escort protein (REP) that then directs it to RAB geranylgeranyl transferase (RabGGT) to receive its prenyl tails (red wavy lines). REP delivers the RAB to its target membrane. Throughout this process, the RAB is GDP-bound. A guanine nucleotide exchange factor (GEF) catalyzes exchange of GDP for GTP to activate the RAB. The GTP-bound RAB interacts with efector proteins that mediate membrane traic in the pathway regulated by its associated RAB. The RAB then interacts with its associated GTPase activating protein (GAP) that catalyzes hydrolysis of GTP to GDP by the RAB. The RAB is then removed from the membrane by guanine nucleotide dissociation inhibitor (GDI) in preparation for the next cycle. Loss-of-function mutations at each of the above steps produce disease phenotypes as indicated by the yellow text boxes[START_REF] Hutagalung | Role of rab gtpases in membrane traic and cell physiology[END_REF].

Figure 4 . 3 RAB

 43 Figure 4.3 RAB GTPases functions in vesicle traicking. Distinct membrane traicking steps that can be controlled by a RAB GTPase and its efectors (indicated in orange). a. An active GTP-bound RAB can activate a sorting adaptor to sort a receptor into a budding vesicle. b. Through recruitment of phosphoinositide (PI) kinases or phosphatases, the PI composition of a transport vesicle might be altered (the conversion of PI-x into PI-y) and thereby cause uncoating through the dissociation of PI-binding coat proteins. c. RAB GTPases can mediate vesicle transport along actin ilaments or microtubules (collectively referred to as cytoskeletal tracts) by recruiting motor adaptors or by binding directly to motors (not shown).d. RAB GTPases can mediate vesicle tethering by recruiting rod-shaped tethering factors that interact with molecules in the acceptor membrane. Such factors might interact with SNAREs and their regulators to activate SNARE complex formation, which results in membrane fusion. e. Following membrane fusion and exocytosis, the RAB GTPase is converted to its inactive GDP-bound form and comes back to the donor membrane[START_REF] Stenmark | Rab gtpases as coordinators of vesicle traic[END_REF].

Figure 4 . 4

 44 Figure 4.4 Diferential regulation of PAR1 constitutive recycling and basal degradation by RAB11A and RAB11B. Unactivated PAR1 is constitutively internalized and recycled back to the cell surface through a RAB11B-dependent pathway, whereas RAB11A regulates basal lysosomal degradation of PAR1 (left panel).In the absence of RAB11A expression (right panel), PAR1 constitutively internalizes, and a marked increase in receptor accumulation in early endosomes is observed, as basal degradation of PAR1 is inhibited. Interestingly, in the absence of RAB11B expression (middle panel), PAR1 exhibits enhanced basal degradation that is blocked by RAB11A and ATG5 depletion, suggesting that the receptor traics through an autophagic pathway before degradation in the autolysosome. These indings suggest that RAB11B and RAB11A serve distinct functions and regulate PAR1 recycling or basal degradation, respectively[START_REF] Grimsey | Recycling and endosomal sorting of protease-activated receptor-1 is distinctly regulated by rab11a and rab11b proteins[END_REF].

Figure 4 . 5

 45 Figure 4.5 Diversity of RAB11 motor protein complexes.RAB11-GTP (green sphere) forms a complex with the microtubule minus-end directed dynein motor protein complex. The interaction is mediated by the interaction of the FIP3 adaptor protein with the DLIC1. The RAB11/protrudin/KIF5 complex represents a RAB11 microtubule plus-end directed motor protein complex. Of note is the adaptor protein protrudin, which belongs to the FYVE zinc inger family of membrane binding proteins; it interacts with RAB11 in its GDP bound form (RAB11-GDP, red sphere). The RAB11-GTP/FIP2/MyoVb complex allows RAB11 vesicles to slide along actin ilaments. The unconventional MyoVb actin motor protein interacts directly with both the FIP2 adaptor and the RAB11 protein[START_REF] Welz | Orchestration of cell surface proteins by rab11[END_REF].

Figure 4 . 6

 46 Figure 4.6 RAB11A/RAB11-FIP2/MYO5B at the level of the recycling endosomes of langerin.Working model for chronological events implicated in the recycling of langerin, from recycling endosomes to the plasma membrane. TIRF temporal characterizations of molecular events that lead to the docking/fusion at the plasma membrane of the recycling vesicles bearing langerin, are summarized in the graphs. Curves represent the average of normalized luorescence levels in a speciic region of the plasma membrane where a double luorescent vesicle is detected, for each time point after temporal registration based on the langerin maximum luorescence level for normalization (Lang-RAB11A n = 15, Lang-RAB11-FIP2 n = 12 and Lang-RAB11-FIP2RBD n = 13) or on RAB11A maximum luorescence level for RAB11A-Myosin Vb and RAB11A-RAB11-FIP2 conditions (n = 10 and 12, respectively). Around 40 frames before and 40 frames after the temporal occurrence of these maximums, are then considered for both luorescence channels in order to obtain values close to baseline of luorescence intensities and are plotted here[START_REF] Gidon | A rab11a/myosin vb/rab11-ip2 complex frames two late recycling steps of langerin from the erc to the plasma membrane[END_REF].

Figure 6 . 3

 63 Figure 6.3 Diferentially expressed genes in muscle-invasive bladder tumors from the TCGA data set.This table provides the results of the diferential expression analysis, performed with the LIMMA method, between supericial tumors associated with altered FGFR3 and without FGFR3 alterations from the TCGA data set. symbol, Gene symbol; adjusted P-value, adjusted p-value using Benjamini et Hochberg procedure; Foldchange, estimate the fold change tumors associated with altered FGFR3 versus without FGFR3 alterations.

Figure 6 . 4

 64 Figure 6.4 RAB25, RAB11A, RAB11B, RAB11FIP2, MYO5B expressions in bladder cancer. Box plots showing gene expression values of RAB25 (A), RAB11A (B), RAB11B (C), RAB11FIP2 (D) and MYO5B (E) in bladder cancer samples from microarray data set (HuExon data set) and from RNA-seq (TCGA data set) comparing tumors carrying mutated and translocated FGFR3 (Altered) and tumors with non-mutated and non-translocated of FGFR3 (Non-altered). Box plots represent the irst and third quartiles and median values; whiskers represent all values from the smallest value up to the largest value. The number of samples for each group was indicated under box plots. Diferential expression analysis was performed by using the LIMMA method. Left: supericial tumors from HuExon data set. Center: muscle-invasive tumors from HuExon data set. Right: muscle-invasive tumors from TCGA dataset. TCGA: The Cancer Genome Atlas; FC: fold change in gene expression of indicated gene between group of altered tumors versus group of non-altered tumors.

Figure 6 . 5

 65 Figure 6.5 RAB25, RAB11A, RAB11B, RAB11FIP2 and MYO5B expressions in normal human urothelial cells and bladder cancer cell lines. Expression values from microarray data set (Afymetrix Human Exon 1.0 ST Array, RMA normalization) of RAB25 (A), RAB11A (B), RAB11B (C), RAB11FIP2 (D), MYO5B (E) and FGFR3 (F) in normal human urothelial cells (NHU) and six bladder cancer cell lines (MGHU3, RT112, TCCSup, UMUC9, UMUC5, HT1376). (G) Protein level of RAB25 was measured by Western Blot in bladder cancer cell lines. Tubulin was used as loading control.

Figure 6 . 6

 66 Figure 6.6 Characteristics of bladder cancer cell lines used in the study.

Figure 6 . 7

 67 Figure 6.7 Depletion of RAB25/RAB11 and their efectors inhibits cell viability. MGHU3 cells (A) and RT112 cells (B) were transfected with diferent siRNA targeting RAB11A, RAB11B, RAB25, RAB11FIP2 and MYO5B. (C) RT112 cells were transfected with diferent siRNA targeting RAB7A/B and RAB21. After 72h transfection, cell viability was determined by the MTT assay. Error bars, SEM from three independent experiments each performed in triplicate. t-test was performed between siRNA targeting indicated genes with control siRNA; *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 6 . 8

 68 Figure 6.8 Eiciency of siRNA silencing in MGHU3 and RT112 cells. (A) In MGHU3 cells, the expression levels of RAB11A, RAB11B, RAB25, RAB11FIP2 and MYO5B after 72h siRNA transfection were veriied by RT-qPCR. Western Blot analysis conirmed the eiciency of siRNA targeting RAB25. (B) In RT112 cells, the expression levels of RAB11A, RAB11B, RAB25, RAB11FIP2 and MYO5B after 72h siRNA transfection were veriied by RT-qPCR; and Western blotting conirmed the eiciency of siRNA targeting RAB25, RAB11A, RAB21 and RAB7A/B. In RT-qPCR, target gene expressions were normalized to TBP level. Tubulin ( -tub) was used as loading control in Western Blot analysis. The igure is representative data from one of three independent experiments.

Figure 6 . 9 Figure 6 .

 696 Figure 6.9 Cell viability after depletion of RAB25/RAB11 and their efectors in other bladder cancer cell lines. TCCSup (A), UMUC9 (B), UMUC5 (C), HT1376 (D) cells were transfected with control siRNA or other indicated siRNAs. After 72h transfection, cell viability was determined by the MTT assay. Error bars, SEM from 2-3 independent experiments each performed in triplicate. t-test was performed between siRNA targeting indicated genes with control siRNA; *, p<0.05; **, p<0.01, ***, p<0.001.

Figure 6 .

 6 Figure 6.11 Efects on cell viability of a combination of RAB11/RAB25 depletion with FGFR3 depletion or treatment of FGFR inhibitor. (A) RT112 cells were transfected with diferent siRNA targeting indicated genes or in combination with siRNA targeting FGFR3. (B) RT112 cells were irst transfected with siRNA targeting RAB25/RAB11 then treated with dimethyl sulfoxide (DMSO) as control or 0.5 M FGFR inhibitor PD173074 for 24h. Cell viability was determined by the MTT assay. In (A) the graph represents the cell viability relative to control siRNA. In (B) the bar plots represent the raw value in MTT assays. t-test was performed, *, p-value<0.05 Error bars, SEM from three independent experiments each performed in triplicate.

Figure 6 . 12 FGFR3Figure 6 .Figure 6 . 14 FGFR3

 6126614 Figure 6.12 FGFR3 localizes to RAB25-and RAB11-positive vesicles. (A) RT112 cells transiently expressing wild type FGFR3-GFP were ixed and stained with anti-EEA1, anti-LAMP1, anti-CD63, anti-TFRC and anti-RAB25. Also, RT112 cells were cotransfected with RAB11A-mcherry. The cells were then ixed and analyzed by 3D deconvolution microscopy. Red signal: EEA1, LAMP1, CD63, TFRC, RAB25 and RAB11A. Green signal: FGFR3. Scale bar, 10 m. (B) RT112 cells transiently expressing WT FGFR3-GFP were ixed and analyzed by 3D deconvolution microscopy. Blue signal: DAPI, green signal: FGFR3. Scale bar, 10 m.(C) Colocalization between FGFR3 and diferent markers was quantiied and Manders' overlap coeicients were calculated using Jacop plugin in ImageJ. The histogram represents the mean ±SEM of two independent experiments. A total of 35-45 cells were analyzed for each condition. t-test was performed. *, p≤0.05; **, p≤0.01.

Figure 6 .

 6 Figure 6.15 Diferential efects of RAB25/RAB11 depletion on FGFR3 downstream signaling pathways. (A) After 72h transfection with indicated siRNAs, RT112 cells were lysed, and proteins were analyzed by Western blotting using antibodies against indicated proteins or phospho-proteins. (B) Phosphorylation of P38 levels was determined after depletion of FGFR3 or RAB11 alone, and after combination of siRNA against RAB25 with either siRNA against FGFR3 or FGFR inhibitor treatment. Tubulin ( -tub) was used as loading control in Western Blot analysis. The blot is a representative data of three independent experiments.

Figure 6 . 16

 616 Figure 6.16 Expression of FGFR3 target genes after RAB25/RAB11 and/or FGFR3 depletion in RT112 cells. RT112 cells were transfected with control siRNA (siCtrl) or indicated siRNAs and cultured for 72h. mRNA extraction and cDNA synthesis by reverse transcription-PCR were performed. The expressions of MYC (A), DUSP6 (B), GATA3 (C) and TIMP2 (D) were determined by RT-qPCR with normalization to TBP gene expression. The graph is data from one experiment.

Figure 6 .

 6 Figure 6.17 Dyngo4a treatment increases the phosphorylation of AKT and ERK1/2 but not P38. (A) RT112 cells were incubated for 30 min at 37 ∘ C with 60 M Dynamin inhibitor Dyngo4a or 0.1% DMSO only in serum-free medium. The cells were then incubated with the same media containing luorescently labeled Alexa-555 transferrin (red) for 45 min at 4 ∘ C. Following two washes, the cells were transferred back to 37 ∘ C for 15 min. The cells were ixed with 4% PFA and stained with DAPI as counterstained, images were taken with luorescence microscopy (objective of 60X). (B)-(D) RT112 cells were treated with 60 M Dyngo4a or 0.1% DMSO for the indicated times. The cells were then lysed and proteins were separated using SDS-PAGE and analyzed by Western blotting with the indicated antibodies. Three blots from three independent experiments were shown. Tubulin ( -tub) was used as loading control.

Figure 7 . 1

 71 Figure 7.1 Generation of wild type and S249C FGFR3-GFP expressing HeLa cells. (A) HeLa cells were transfected with WT or S249C FGFR3 tagged with GFP at C-terminal (WT FGFR3-GFP, S249C FGFR3-GFP) or with mock GFP plasmid (Mock-GFP). 24h after transfection, the cells were lysed and analyzed by Western blotting with anti-FGFR3. Tubulin was used as loading control. (B) Hela cells transiently expressing WT FGFR3-GFP or S249C FGFR3-GFP were ixed and analyzed by 3D deconvolution microscopy. Scale bar, 10 m. (C) HeLa cells were transfected with WT FGFR3-GFP (wt) or S249C FGFR3-GFP (S249C)for 24h, then treated with DMSO alone (0.1%) as control or FGFR inhibitor PD173074 (1 M) for 15 minutes (15m) and 1 hour (1h). The cells were then lysed and immunoprecipated with anti-FGFR3. In addition, the cell lysate from HeLa expressing WT FGFR3-GFP was incubated with IgG control (IgG). The protein complex was then eluted and analyzed by Western blotting using anti-phosphotyrosine (anti-PY) or anti-FGFR3. Flow through of immunoprecipitation was also loaded to verify the binding eiciency of antibody to antigen.

7. 2

 2 Results 95

7.2. 3 FGFR3EEA1S249CFigure 7 . 2

 372 Figure 7.2 Subcellular localization of WT and S249C FGFR3. HeLa cells transiently expressing WT or S249C FGFR3-GFP were ixed and stained with anti-EEA1, anti-TFRC, anti-LAMP1 and anti-CD63. HeLa cells were also co-transfected with a RAB11A-mcherry with either WT FGFR3-GFP or S249C FGFR3-GFP. The cells were then analyzed by 3D deconvolution microscopy. Red signal: EEA1, LAMP1, CD63, TFRC and RAB11A. Green signal: FGFR3. Scale bar, 10m.

Figure 7 . 3 Figure 7 . 4 WT

 7374 Figure 7.3 Subcellular localization of WT and S249C FGFR3. Colocalization between WT FGFR3 (A) or S249C FGFR3 (B) with diferent markers shown in Figure 7.2 was quantiied and Manders' overlap coeicients were calculated, t-test was performed. The histogram represents the mean ± SEM of three independent experiments. A total of 55-65 cells were analyzed for each condition. (C) Manders' coeicients were compared between two WT and S249C forms of FGFR3, t-test was performed. *, p<0.05; **, p<0.01; ***, p<0.001; ns, non-signiicant.

Figure 7 . 5 S249C

 75 Figure 7.5 S249C FGFR3 accumulates in TFRC-positive structures in RAB11-depleted cells. (A) HeLa cells were transfected with control siRNA (siCtrl) or siRNA targeting RAB11A/B followed by transfection with S249C FGFR3-GFP. The cells were then ixed and stained with anti-EEA1, anti-TFRC, anti-LAMP1 and anti-CD63. Red signal: EEA1, LAMP1, CD63 and TFRC. Green signal: FGFR3. Scale bar, 10m. (B) Knockdown eiciency was veriied by RT-qPCR, normalized to TBP expression.

Figure 7 . 6 FGFR3

 76 Figure 7.6 FGFR3 accumulated in TFRC-positive structures in RAB11-depleted cells. Colocalization between WT FGFR3 (A) or S249C FGFR3-GFP (B) with diferent markers shown in Figure 7.4 and Figure 7.5 was quantiied and Manders' overlap coeicients were calculated. The histogram represents the mean ± SEM of two independent experiments. A total of 35-45 cells were analyzed for each condition. t-test was performed, *, p<0.05; **, p<0.01, ns, non-signiicant.

7. 3 Figure 7 . 7

 377 Figure 7.7 Signaling of WT and S249C FGFR in HeLa expressing FGFR3-GFP. (A) Cell lysates from HeLa cells transiently expressing WT or S249C FGFR3-GFP or mock-GFP plasmid and HeLa cells nontransfected were analyzed by Western blotting with anti-FGFR3, anti-phospho-P38, anti-phospho-AKT and anti-phospho-ERK1/2. (B) Cell lysates from RAB11-depleted HeLa cells transfected with WT or S249C FGFR3-GFP were analyzed by Western blotting using anti-phospho-AKT, anti-phospho-P38, anti-phospho-ERK1/2, anti-RAB11A. Tubulin was used as loading control.

Figure 8 . 1

 81 Figure 8.1 Scatter plot of the protein abundance ratios against the intensities of the identiied proteins. Scatter plot of the abundance ratios between WT FGFR3-GFP versus GFP control, plotted against the intensities for each protein identiied. The square corresponds to proteins that are not signiicantly enriched. The red cross indicates signiicant protein abundance diferences between WT FGFR3-GFP versus GFP, p-value <0.05 for Experiment 1 (A), and if the abundance ratios between WT FGFR3-GFP versus GFP ≥4 for experiment 2 (B).

8. 3

 3 Results 109 poorly explored.

Figure 8 . 3

 83 Figure 8.3 Network 1. Continuous line: direct interaction; Dash line: indirect interaction; Line with arrowhead: protein A acts on protein B. Gray node: protein identiied in mass spectrometry; white node: protein from IPA database.

Figure 8 . 4 8 . 3 111 Figure 8 . 5 Network 3 .

 8483111853 Figure 8.4 Network 2. Continuous line: direct interaction; Dash line: indirect interaction; Line with arrowhead: protein A acts on protein B. Gray node: protein identiied in mass spectrometry; white node: protein from IPA database.

Figure 8 . 6 Network 4 .

 864 Figure 8.6 Network 4. Continuous line: direct interaction; Dash line: indirect interaction; Line with arrowhead: protein A acts on protein B. Gray node: protein identiied in mass spectrometry; white node: protein from IPA database.

8. 3 113 Figure 8 . 7 Network 5 .

 3113875 Figure 8.7 Network 5. Continuous line: direct interaction; Dash line: indirect interaction; Line with arrowhead: protein A acts on protein B. Gray node: protein identiied in mass spectrometry; white node: protein from IPA database.

Figure 9 .

 9 Figure 9.1 FGFR3 traicking and signaling in bladder cancer carrying altered FGFR3. Green arrow: traicking pathway, blue arrow: signaling pathway. Continuous line: pathway was proven in our study or other studies. Dash line: proposed pathway. FGFR3: Fibroblast Growth Factor Receptor 3, FGFR3-TACC3: gene fusion between FGFR3 and Transforming Acidic Coiled-Coil containing protein 3, TFRC: Transferrin Receptor, DUSP6: Dual Speciicity Phosphatase 6, ETS2: V-Ets Avian Erythroblastosis Virus E26 Oncogene Homolog 2, TTP: Tristetraprolin, PUM2: Pumilio RNA-Binding Family Member 2, HIF1 : Hypoxia Inducible Factor 1 Alpha Subunit.

Figure 2.11 Acquired and heritable mutations in FGFs and FGFRs in malignancy

			2.5 Alterations of FGFR3 in bladder cancer 31
	Gene Name Mutation	Type of Cancer
	(a) Contributions of FGFs to malignancy (in vivo)
	FGF1	Amplification	Ovarian cancer
	FGF2	Over expression	Bladder cancer, Prostate cancer, Small cell lung carcinoma,
			Melanoma, Hepatocellular carcinoma
	FGF3	Amplification	Breast cancer
	FGF4	Amplification	Breast cancer
	FGF5	Over expression	Glioblastoma
	FGF6	Over expression	Prostate cancer
	FGF7	Over expression	Lung adenocarcinoma
	FGF8	Over expression	Breast cancer, Prostate cancer, Hepatocellular carcinoma,
			Colorectal cancer
	Fgf9	Frameshift/missense/	Colorectral and endometrial carcinomas
		nonsense mutation
		Over expression	Non small cell lung cancer
	FGF10	Over expression	Breast carcinomas, Prostate cancer
	FGF15/19	Over expression	Prostate cancer, Hepatocellular carcinoma
	FGF16	Over expression	Ovarian cancer
	FGF17	Over expression	Prostate cancer, Hepatocellular carcinoma
	FGF18	Over expression	Hepatocellular carcinoma
	FGF22	Knockout	Suppresses skin papilloma (in mice)
	FGF23	Polymorphism	Increased risk of prostate cancer
	(b) Contributions of FGFRs to malignancy (in vivo)
	FGFR1	Amplification	Small cell lung cancer, Squamous cell lung cancer, Breast cancer,
			Ovarian cancer, Pancreatic ductal adenocarcinoma, Tongue
			squamous cell carcinoma
		Missense mutation	Melanoma, Pilocytic astrocytoma
		Translocation	Leukemia, Lymphoma, Alveolar rhabdomyosarcoma, Glioblastoma,
			Myeloproliferative syndrome (fusion with CUX1, FGFROP2, FIM,
			RANBP2/NUP358, SQSTM1, TRP, ZNF198)
		Over expression	Glioblastoma
	FGFR2	Amplification	Gastric cancer, Breast cancer
		Missense mutation	Endometrial carcinoma, Gastric cancer
		Translocation	Cholangiocarcinoma
	FGFR3	Missense mutation	Gastric cancer, Colorectal cancer, Breast cancer, Endometrial
			carcinoma, Urothelial carcinoma, Bladder tumor, Skin tumor,
			Myeloma
		Mis-localization	Brest cancer
		Translocation	Myeloma, Squamous cell lung cancer, Bladder cancer,
			Glioblastoma, Lymphoma
		Over expression	Breast cancer, Colon cancer (FGFR3c)
	FGFR4	Missense mutation	Rhabdomyosarcoma, Adenoid cystic carcinoma, Breast Cancer
			(resistance to adjuvant therapy)
		Over expression	Ovarian cancer, hepatocellular carcinoma

[START_REF] Ornitz | The ibroblast growth factor signaling pathway[END_REF]

.

Table 4 .1 A summary of RAB25 with dysregulated expression level in human cancers

 4

	Cancer

1 Diferentially expressed genes in supericial bladder tumors from the HuExon data set

  .6. RAB11A, RAB11B and RAB11FIP2 were expressed at about the same levels in the NHU cells and

	rank Figure 6.6.2 Results 71 symbol adjusted P-Value Foldchange 1 PIK3R1 0,000188947 1,539968736 2 RIMS2 0,026997051 1,474632875 3 RAB38 0,027841026 1,405831019 4 TBC1D4 0,001983594 1,401916598 5 TRAPPC1 3,13E-06 1,379190849 6 CASP1 0,000134854 1,371910077 7 SDC1 8,58E-05 1,370668009 8 SYTL1 6,28E-06 1,351344304 9 ARL6IP5 0,0041291 1,31544224 10 MADD 0,001546274 1,259644611 11 RAB13 2,50E-05 1,241570712 12 ANKFY1 0,001058843 1,234284217 13 ARHGAP1 0,012123341 1,222363577 14 RAB25 0,005958683 1,212426267 15 ATG5 0,002837457 1,202575817 16 RAB40A 0,000820313 1,196018227 17 MICAL1 0,015962224 1,195739034 18 VPS52 0,003679771 1,183041163 19 RAB3GAP1 0,0041291 1,178574247 20 RAB19 0,039223502 1,168075099 21 GCC2 0,035364187 1,164983868 22 GOPC 0,003016732 1,164557379 23 RAB4A 0,0406342 1,152543272 24 VPS11 0,028589834 1,14471658 25 VPS39 0,012902615 1,143180691 26 CEP290 0,03785452 1,127132044 27 TSC2 0,035364187 1,118595899 28 RPH3AL 0,012902615 1,116185441 29 ATG16L1 0,042029964 1,114190956 30 STX4 0,042458806 1,097606414 31 TBC1D17 0,039223502 1,092332195 32 CTBP1 0,037665171 1,091766709 33 TBC1D3B 0,0041291 1,070800851 34 GNB1 0,026313005 0,914986003 35 RAB8A 0,035364187 0,893828255 36 ITGB1 0,004543994 0,888062689 37 RAB5A 0,018640413 0,886781259 38 RAB1A 0,012329798 0,884933748 39 GNG2 0,014161156 0,879689273 40 ODF2 0,037535659 0,874997826 41 YWHAQ 0,003236383 0,869812089 42 GOLGA2 0,026869759 0,86060278 superficial HuExon data set 1 SDC1 0,000675877 2,407568583 2 PIK3R1 0,000369297 1,858906697 3 RAB25 0,036350205 1,726434901 4 RAB19 0,000968118 1,670995335 5 SYTL1 4,18372E-06 1,587077481 6 ALS2CL 0,047203843 1,578594711 7 RAB11A 0,019349988 1,338720002 8 VPS35 0,027367285 1,283480731 9 DENND1A 0,019349988 0,759125844 10 ERC1 0,019349988 0,757020264 11 LEPRE1 0,042189453 0,713209774 12 GNG2 0,008947891 0,678566508 13 MYO5A 0,024060497 0,633283592 14 MICAL2 0,032321388 0,631038863 15 STXBP1 0,006117843 0,503061626 rank symbol adjusted P-Value Foldchange invasive HuExon data set

. This table provides results of the diferential expression analysis, performed with the LIMMA method, between supericial tumors associated with altered FGFR3 and without FGFR3 alterations from the HuExon data set. symbol, Gene symbols; adjusted P-value, adjusted p-value using Benjamini et Hochberg procedure; Foldchange, estimate the fold change tumors associated with altered FGFR3 versus without FGFR3 alterations.

Figure 6.2 Diferentially expressed genes in invasive bladder tumors from the HuExon data set.

  This table provides the results of the diferential expression analysis, performed with the LIMMA method, between invasive tumors associated with altered FGFR3 and without FGFR3 alterations from the HuExon data set. symbol, Gene symbols; adjusted P-value, adjusted p-value using Benjamini et Hochberg procedure; Foldchange, estimate the fold change tumors associated with altered FGFR3 versus without FGFR3 alterations.

	31 MADD 0,000106257 1,289220111	30 TBC1D4 0,02146894 1,298624327	29 CTBP1 6,13E-06 1,309043196	28 RAB3A 0,00986944 1,334234891	27 RIN1 0,043337692 1,353507076	26 RAB40C 0,000553367 1,354286464	25 MICALL2 0,001861668 1,369498084	24 TRAPPC5 0,000489903 1,389143502	23 CD2AP 0,000251303 1,405731433	22 TRAPPC1 4,04E-06 1,410081118	21 RAB11FIP1 0,023901495 1,415157718	20 CEP290 8,16E-05 1,43464028	19 RILP 0,000780461 1,446714548	18 RABEP2 5,11E-05 1,455065593	17 NDRG1 0,018729337 1,487005447	16 RAB11A 4,75E-08 1,501377038	15 GAPDH 4,04E-06 1,528420871	14 SYTL4 0,00019751 1,569350604	13 RAB19 0,018729337 1,606288906	12 TBC1D3B 0,000148969 1,717469353	11 ICA1 0,000695707 1,754847848	10 ALS2CL 0,000128078 1,898650103	9 RAB17 0,000449901 1,930939288	8 RPH3AL 6,10E-08 2,091117987	7 RAB15 4,75E-05 2,12548073	6 RAB26 0,000553367 2,17754529	5 SDC1 0,000148969 2,250560098	4 RAB25 0,001714843 2,26741529	3 OTOF 0,001232766 2,347246647	2 MLPH 1,26E-05 2,553810413	1 SYTL1 6,03E-09 3,055054651	rank symbol adjusted P-Value Foldchange invasive TCGA data set (1)
	62 RAB21 0,044042883 1,136280054	61 RAB1B 0,017681472 1,136838567	60 GRIPAP1 0,010626637 1,140050803	59 RUFY1 0,014504887 1,149297274	58 VPS29 0,013538382 1,150845307	57 RAB10 0,018287843 1,170261497	56 RAB5B 0,007296844 1,175571257	55 ARL6IP5 0,035469788 1,179433135	54 TRAPPC9 0,016600318 1,180206579	53 TRAPPC2 0,018729337 1,180216892	52 CTNNA1 0,001364181 1,184801045	51 GAS8 0,018986795 1,188221194	50 RAB11B 0,003513539 1,199246129	49 ZFYVE27 0,002873326 1,20117308	48 TBC1D17 0,004602969 1,20274616	47 HPS1 0,004307102 1,206976517	46 VPS39 0,000148969 1,206983803	45 VPS35 0,002250204 1,207450326	44 RAC1 0,001113514 1,207925213	43 TBC1D2B 0,007378603 1,210380785	42 HPS4 0,008233756 1,211192444	41 EVI5L 0,009116263 1,21533956	40 TSC2 0,000148969 1,215465793	39 NSF 0,003913507 1,225143933	38 VPS33B 1,76E-05 1,234418843	37 RAB4B 0,008717868 1,237667375	36 RAB13 0,003296464 1,240673331	35 RAB4A 0,000148969 1,240887255	34 MICALL1 0,02308131 1,245596636	33 RAB24 0,001304836 1,261929313	32 VPS52 4,04E-06 1,26625043	rank symbol adjusted P-Value Foldchange invasive TCGA data set (2)
	93 GAPVD1 4,83E-11 0,687028861	92 VCL 0,000211701 0,68984344	91 PRKAR2A 0,021892483 0,691717917	90 RAB6B 0,033459137 0,699671227	89 BICD2 0,000541115 0,718060226	88 OSGIN2 0,000116822 0,728566905	87 ODF2 1,15E-06 0,731192327	86 RNF115 7,50E-05 0,738441745	85 TBXA2R 0,049139422 0,740118653	84 ANKRD27 0,000110252 0,743358893	83 PIK3CB 0,000591303 0,775232188	82 LEPRE1 0,023589339 0,778233245	81 RIN2 0,035547259 0,788442109	80 YWHAQ 0,001701333 0,793547178	79 INPP5B 0,000539707 0,795782582	78 GOLGA2 0,000148969 0,796544211	77 RABGAP1 0,00047591 0,798958174	76 ZFYVE20 0,002728235 0,804838714	75 DYNC1LI1 0,000239554 0,807240949	74 RAB9A 0,008233756 0,81045821	73 SMCHD1 0,016977318 0,811077214	72 RABEPK 0,004532116 0,811139799	71 WDR44 0,032857762 0,812213112	70 ITGB1 0,015449217 0,828671019	69 MICAL3 0,031332417 0,830854052	68 ACAP2 0,007378603 0,832116946	67 RAB14 0,001232766 0,84014812	66 INPP5E 0,035569027 0,841118357	65 DCTN1 0,001693511 0,849958215	64 RAB33B 0,045542105 0,853249545	63 GOLGA1 0,001701333 0,853984014	rank symbol adjusted P-Value Foldchange invasive TCGA data set (3)
			122 SNAP25 6,95E-07 0,23055123	121 CAV1 4,04E-06 0,328798108	120 RAB3IL1 2,38E-06 0,352730821	119 RAB42 3,18E-07 0,404269661	118 RAB39 0,000110276 0,408795722	117 STXBP1 2,10E-05 0,413921253	116 LRRK2 0,000325562 0,415287428	115 GNAL 0,003008379 0,459418955	114 GNG2 3,11E-05 0,460572151	113 RAB39B 0,000823133 0,464478202	112 RAB30 1,21E-06 0,48581967	111 RAB33A 8,02E-05 0,512864267	110 RAB31 0,000109006 0,517107345	109 ITGA5 0,000576308 0,52533673	108 TBC1D10C 0,010903306 0,532239316	107 RAB3C 0,012479574 0,533856896	106 RAB23 0,000109006 0,543862586	105 RIMS1 0,026595181 0,551561059	104 MICAL2 4,88E-05 0,561912578	103 WDR38 0,009116263 0,565596042	102 PEX5L 0,009116263 0,574726444	101 PTGIR 0,001671952 0,576585626	100 RUSC2 0,000469632 0,593093048	99 MYO5A 0,000656026 0,594465713	98 SYTL3 0,001364181 0,603257573	97 DENND5A 1,15E-06 0,613256471	96 ERC1 2,08E-06 0,62087021	95 RABGAP1L 4,75E-05 0,628380452	94 DENND1A 2,37E-07 0,659950834	rank symbol adjusted P-Value Foldchange invasive TCGA data set (4)

Table 6 .1 List of siRNA used in the study.

 6

	siRNA Control siRNA CGUACGCGGAAUACUUCGATT (sens) Sequence	Concentration as indicated in igures
	RAB11A no6	UCGAAGUAUUCCGCGUACGTT (anstisens) AAAUGAGUUUAAUCUGGAATT (sens)	10nM
	RAB11B no4	UUCCAGAUUAAACUCAUUUCG (anstisens) AGUUCAACCUGGAGAGCAATT (sens)	10nM
	RAB25 no3	UUGCUCUCCAGGUUGAACUCG (antisens) GGAACUGAGGAAGAUUAUATT (sens)	10nM
	UAUAAUCUUCCUCAGUUCCAT (antisens) RAB11FIP2 no6 CCUGGUGGGUCUGGAUAAATT (sens)	20nM
	MYO5B no7	UUUAUCCAGACCCACCAGGGA (antisens) GCAUUAGUUUGAUUCCCAATT (sens)	20nM
	FGFR3	UUGGGAAUCAAACUAAUGCTG (antisens) CCUGCGUCGUGGAGAACAATT (sens)	4nM
		UUGUUCUCCACGACGCAGGTG (antisens)

Table 6 .2 List of primers and probes used for RT-qPCR.

 6

	Gene (Probe) RAB11A (31) RAB11B (62) RAB25 (1) RAB11FIP2 (59) GCAATGAAGACCTCAGGAAAA Forward primer CTGCACCTTTGGCTTGTTTT CGACGAGTACGACTACCTATTCAA AACTCGTTGCGGGTGAAG Reverse primer CAGGGCAGTTCCTACAGATGA GCTGCTGTCAAGGCTCAGAT CCCACTGCACCACGATAGTA CTTCATAGGTCAGACTACGATACCC MYO5B (49) CTCCAACAAGGAGCACAAGA CAGGGTCTCCATGAGCAGAT MYC (34) CACCAGCAGCGACTCTGA GATCCAGACTCTGACCTTTTGC DUSP6 (66) CGACTGGAACGAGAATACGG AATGTACCAAGACACCACAGTTCT TIMP2 (43) GAAGAGCCTGAACCACAGGT CGGGGAGGAGATGTAGCAC GATA3 (71) CTCATTAAGCCCAAGCGAAG TCTGACAGTTCGCACAGGAC

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