The Hedgehog receptor Patched (Ptch) is a well-studied tumor suppressor. Mutations in Ptch have been linked to mis-regulation of stem cell proliferation and tumorigenesis in numerous contexts. To study the role of Ptch function during retinal development and homeostasis, I analyzed retinal growth and patterning in the embryonic and post-embryonic (juvenile) zebrafish ptc2 mutant line. ptc2 deficiency in zebrafish results in an expansion of the stem/progenitor population of the ciliary marginal zone (CMZ), as well as ectopic proliferation within the neural retina at juvenile stages. ptc2-/- mutants also possess vitreo-retinal abnormalities that appear to be embryonic in origin. These phenotypes are similar to the ocular abnormalities previously reported in human patients suffering from Basal Cell Naevus Syndrome (BCNS), a disorder that has been linked to mutations in the human PTCH gene (the orthologue of the zebrafish ptc2), and point to the utility of the ptc2 mutant line as a model for the study of BCNS-related ocular pathologies. In addition, peripheral retinal dysplasias that include ectopic neuronal clusters and disrupted lamination were observed at later, juvenile stages. It has been previously proposed that retinal over-proliferation might contribute to retinal dysplasias observed in the post-natal Ptch1 /- mice (an established model for BCNS); however, this potential relationship has yet to be established experimentally. I demonstrated that a population of ectopically proliferating cells give rise to the ectopic neuronal clusters in the INL of ptc2-/- mutants and established ectopic proliferation as the likely cellular underpinning of retinal dysplasia in juvenile ptc2-/- mutants. / text

Hedgehog is a lipid-linked morphogen that is carried on lipoprotein particles and that regulates both patterning and proliferation in a wide variety of vertebrate and invertebrate tissues. Hyperactivity of Hedgehog signaling causes numerous forms of cancer. Hedgehog acts by binding to its receptor Patched, relieving the suppression of Smoothened and initiating Smoothened signaling. The mechanism by which Patched represses Smoothened has been unclear, but correlates with reduced Smoothened levels on the basolateral membrane. The structural homology of Patched with the Niemann-Pick-Type C1 protein and bacterial transmembrane transporters suggests that Patched might regulate lipid trafficking to repress Smoothened. However, no endogenous lipid regulators of Smoothened have yet been identified, nor has it ever been shown that Patched actually controls lipid trafficking. This work shows that, in Drosophila melanogaster, the Sterol-Sensing Domain of Patched regulates Smoothened trafficking from Patched-positive endosomes. Furthermore, it demonstrates that Patched recruits internalized lipoproteins to Patched-positive endosomes. Thereby, Patched regulates the efflux of specific lipoprotein-derived lipids from this compartment via its Sterol-Sensing Domain and utilizes these lipids to destabilize Smoothened on the basolateral membrane. We propose that Patched normally promotes Smoothened degradation and subsequently downregulates its activity by changing the lipid composition of endosomes through which Smoothened passes. For this purpose, Patched utilizes a specific lipid – possibly a modified sterol or sphingolipid – derived from lipoproteins. Further, we suggest that the presence of Hedgehog on lipoprotein particles inhibits utilization of their lipids by Patched.

<p>Studying the early stages of cancer can provide important insight into the molecular basis of the disease. In many human cancers, such as prostate, pancreatic, and colon cancer, a pre-neoplastic, or intermediate, stage of the disease has been identified. The pre-neoplastic stage is presumed to be a transition during which normal cells undergo malignant transformation. However, the link between the pre-neoplastic cells and end-stage disease has never been formally established. To investigate the fate of such cells, the patched (ptc) mutant mouse, a model for the brain tumor medulloblastoma was used. Pre-neoplastic cells (PNCs) are found in most ptc mutants during early adulthood, but only 15% of these animals develop tumors. Although PNCs are found in mice that develop tumors, the ability of PNCs to give rise to tumors has never been demonstrated directly, and the fate of cells that do not form tumors remains unknown. Genetic fate mapping and orthotopic transplantation provided definitive evidence that PNCs give rise to tumors and showed that the predominant fate of PNCs that do not form tumors is differentiation. Moreover, N-myc, a gene commonly amplified in medulloblastoma, can dramatically alter the fate of PNCs, preventing differentiation and driving progression to tumors. Importantly, N-myc allows PNCs to grow independently of hedgehog signaling, making the resulting tumors resistant to hedgehog antagonists. These studies provide the first direct evidence that PNCs can give rise to tumors, and demonstrate that identification of genetic changes that promote tumor progression is critical for designing effective therapies for cancer.</p> / Dissertation

No description available.

Hedgehog (HH) signaling controls renal development. Mutations in PTC1, the HH receptor, cause cancer in non-renal tissues. We hypothesized that constitutively active HH signaling is deleterious to renal development in mice with PTC1 deficiency targeted to the metanephric mesenchyme (MM)(Rarb2-Cre;Ptc1 loxP/-, termed Ptc1 mutants). Increased HH signaling in MM of mutant mice was confirmed by qRT-PCR for Ptc1. A decrease in NCAM-positive nephrogenic precursors at E13.5 and WT1-positive glomeruli at E18.5 was found. Increased cortical expression of Foxd1 was observed. At E13.5, a cluster of ectopic cells expressing Raldh2, Ptc2 and Bmp4 accumulated at the presumptive uretero-pelvic junction (UPJ). Magnetic resonance imaging demonstrated an increase in pelvic volume. Constitutive expression of GLI3 repressor via the Gli3Δ699 allele in Ptc1 mutants increased nephron number comparable to wild type mice and decreased pelvic volume compared to Ptc1 mutants. Thus repression of HH activity is required for proper nephrogenesis and patterning of the UPJ.

Hedgehog (HH) signaling controls renal development. Mutations in PTC1, the HH receptor, cause cancer in non-renal tissues. We hypothesized that constitutively active HH signaling is deleterious to renal development in mice with PTC1 deficiency targeted to the metanephric mesenchyme (MM)(Rarb2-Cre;Ptc1 loxP/-, termed Ptc1 mutants). Increased HH signaling in MM of mutant mice was confirmed by qRT-PCR for Ptc1. A decrease in NCAM-positive nephrogenic precursors at E13.5 and WT1-positive glomeruli at E18.5 was found. Increased cortical expression of Foxd1 was observed. At E13.5, a cluster of ectopic cells expressing Raldh2, Ptc2 and Bmp4 accumulated at the presumptive uretero-pelvic junction (UPJ). Magnetic resonance imaging demonstrated an increase in pelvic volume. Constitutive expression of GLI3 repressor via the Gli3Δ699 allele in Ptc1 mutants increased nephron number comparable to wild type mice and decreased pelvic volume compared to Ptc1 mutants. Thus repression of HH activity is required for proper nephrogenesis and patterning of the UPJ.

Nous avons récemment démontré que le récepteur du morphogène Hedgehog, Patched, qui est exprimé dans de nombreux cancers, est un transporteur de multiples drogues qui contribue à la résistance des cellules cancéreuses à la chimiothérapie. Le criblage d'une banque de molécules nous a permis d'identifier deux molécules qui inhibent l'activité d'efflux de doxorubicine de Patched. Nous avons montré que ces molécules renforcent les effets cytotoxiques, proapoptotiques, antiprolifératifs et anticlonogéniques de la doxorubicine sur les cellules de cancer de la glande surrénale (surrénalome) qui expriment de façon endogène Patched. De plus, nous avons observé que l’ajout de la molécule P375 au traitement à la doxorubicine inhibe le développement des tumeurs chez des souris ayant reçu une xénogreffe de cellules de surrénalome de façon plus significative que la doxorubicine seule. Nos résultats suggèrent que l'utilisation d'un inhibiteur de l'activité d'efflux de drogues de Patched en association avec la doxorubicine est une option thérapeutique prometteuse pour le surrénalome, et très probablement pour d'autres cancers exprimant Patched. Nous avons découvert qu'une petite fraction seulement des cellules de la lignée de surrénalome exprime Patched au niveau de la membrane plasmique (cellules PM-Patched). Les cellules PM-Patched sont plus résistantes à la doxorubicine, et présentent une expression plus élevée de Patched mais aussi de la protéine ABCG2. ABCG2 étant un marqueur de cellules souches cancéreuses (CSC), nous pensons que les cellules PM-Patched pourraient être des CSC.D'autres expériences sont nécessaires pour valider cette hypothèse. / We recently demonstrated that the Hedgehog receptor Patched, which is expressed in many recurrent and metastatic cancers, is a multidrug transporter contributing to chemotherapy resistance. The screening of a chemical library allowed us identifying two molecules which inhibit the doxorubicin efflux activity of Patched. We showed that these molecules enhance the cytotoxic, proapoptotic, antiproliferative and anticlonogenic effects of doxorubicin on adrenocortical carcinoma (ACC) cells which endogenously express Patched. Moreover, we reported that the addition of the drug-like molecule P375 to doxorubicin treatment prevents the development of xenograft ACC tumours in mice much more significantly than the doxorubicin alone. Our results suggest that the use of an inhibitor of Patched drug efflux activity in combination with doxorubicin is a promising therapeutic option for ACC and most likely for other Patched-expressing cancers. We discovered that only a small fraction of the ACC cell line expressed Patched at the plasma membrane (PMPatched cells). We observed that these cells are more resistant to doxorubicin treatment than ACC cells that express Patched only in intracellular compartments. Moreover, we estimated that PMPatched cells have higher expression of Patched but also of ABCG2/BCRP proteins. Based on the fact ABCG2/BCRP is a cancer stem cell (CSC) marker and that Hedgehog signaling is involved in maintenance of CSC, we think that PM-Patched cells could be CSCs. More experiments are needed to confirm this hypothesis.

Parmi les voies de signalisation, la voie hedgehog (HH) intervient dans la formation de la polarité segmentaire. Si elle est défectueuse, elle entraine plusieurs malformations. De nombreux cancers présentent une suractivation de cette voie. La voie HH activée par la fixation du ligand HH sur le récepteur Patched (hPtc) et fait intervenir plusieurs partenaires cytoplasmiques dont Supressor of Fused (SUFU).Peu de données moléculaires et structurales sont disponibles pour cette voie et pourtant, ces données sont nécessaires pour comprendre sans ambiguïté son fonctionnement. De plus, la voie HH a été proposée comme pouvant être la cible de traitements chimio thérapeutiques mais, la protéine hPtc est impliquée dans l’efflux des drogues anticancéreuses. Une inhibition de hPtc par la fixation de son ligand entraine l’inhibition de l’efflux de drogues. Néanmoins, le site de fixation de HH sur son récepteur n’a pas encore été déterminé.Durant cette thèse, les travaux effectués ont permis l’étude structurale de la protéine hPtc notamment la détermination du site de fixation de HH. Dans un deuxième volet de cette thèse, j’ai effectué des études structurales de certaines protéines SUFU.Dans un premier temps, je me suis concentrée sur les domaines extracellulaires de hPtc qui ont été décrits comme nécessaires pour la fixation du ligand HH. J’ai cloné une protéine chimère constituée de ces deux domaines liés par le lysozyme du phage T4 (hPtcD1D2). Cette construction a été exprimée dans la bactérie E.coli. Les conditions d’expression testées permettent d’obtenir la protéine sous forme de corps d’inclusion dans le cytoplasme de la bactérie. Dans un deuxième temps, j’ai cloné la protéine dans un vecteur d’expression en levure. De manière concomitante, j’ai exprimé la protéine hPtc tronquée de ses régions N et C terminales (hPtcΛNΛC). Ce sont des régions intrinsèquement désordonnées qui ne permettraient pas une bonne cristallisation de la protéine. L’expression a été effectuée dans la levure. La solubilisation de cette protéine membranaire est en cours d’expérimentation.Ce travail a permis de poser les bases de l’expression de hPtcD1D2 et de hPtcΛNΛC. Ceci va notamment permettre la surexpression de la protéine et sa cristallisation afin de déterminer sa structure 3D et de caractériser le site de fixation de son ligand.Enfin, j’ai entrepris des études structurales des protéines SUFU. Un nouveau site de fixation du Zn a été caractérisé. En effet, après purification de la protéine, j’ai effectué des mesures d’affinité à l’aide d’un composé colorimétrique, le PAR et des expériences de spectroscopie d’émission atomique dans lesquelles j’ai fait varier le pH et la concentration en Zn. Ainsi, j’ai pu déterminer que SUFU a une affinité nanomolaire pour le Zn meilleure à pH 8 qu’à pH 6,5. La fixation du Zn se ferait donc sur un site basique. La structure de SUFU a été publiée en 2013 par deux équipes, je me suis inspirée des conditions de cristallisation de ces deux articles, pour cristalliser SUFU en présence de Zn. Les expériences de dichroïsme circulaire ont permis d’affirmer que ces protéines sont organisées en hélices α et en feuillets β. De plus, grâce à la diffusion des rayons X aux petits angles, j’ai pu déterminer que dSUFU, hSUFU et zSUFU n’ont pas la même conformation en solution. Alors que SUFU de drosophile est un monomère globulaire, les protéines humaine et de poisson zèbre seraient plutôt allongées et dimériques. La région N-terminale potentiellement impliquée dans la dimérisation de hSUFU a été tronquée et hSUFUΛ30 présente des différences d’état d’oligomérisation. / The hedgehog (HH) signalling pathway is involved in the segmentary polarity formation. A dysfunction of this pathway is involved in several malformations. Many cancers are caused by an overactivation of this pathway. The HH signalling pathway is activated by the binding of HH on the receptor Patched (hPtc) and included many cytoplasmic partners such as Suppressor of Fused (SUFU). Few molecular and structural data are available on this pathway even if these data are important to fully understand the pathway functioning. Furthermore, the HH signalling pathway maybe be the target of chemotherapy treatments. However, hPtc is involved in drugs efflux. Inhibition of hPtc by the binding of its ligand HH may lead to this efflux inhibition. Yet, the binding site of HH on its receptor hPtc is not yet determined.During this thesis, the structural study of hPtc have been engaged especially the study of the binding site of HH. On the second hand, I have structurally studied some SUFU proteins.First of all, I have expressed the extracellular domains of hPtc. These domains have been described as necessary for HH binding. I have cloned a chimeric protein made by the extracellular domains of hPtc associated with the lysozyme T4 (hPtcD1D2). This protein have been expressed in the E.Coli bacteria. The protein expressed in inclusion bodies in the cytoplasm of the bacteria. In the other hand, I have cloned the protein in a yeast expression vector. Part of this, I have also expressed the protein hPtc without its N and C terminus regions (hPtcNC). These regions are intrinsically disrupted. They may lead to crystallization problems. The protein has been expressed in yeast.This work permits to expressed hPtcD1D2 and hPtcΛNΛC. This will lead to the expression of the protein and its crystallisation in order to determine its 3D structure and to characterize its ligand binding site.Finally, I structurally studied the protein SUFU. A novel Zn binding site has been characterized. In fact, after the protein purification, I have made affinity measures using a colorimetric compound, PAR. I also performed spectroscopic experiments in which I varied the pH and the Zn concentration. I determined the SUFU has a nanomolar affinity for the Zn best at pH 8 than pH 6.5. Indeed, the Zn binding site may be basic.The SUFU 3D structure has been published in 2013 by two teams. Inspired by their crystallization conditions, I crystallized SUFU with Zn. Circular dichroism experiments permitted to know that the proteins are organized in  helices and  sheets. Moreover, small angles X ray spectroscopy experiments show that dSUFU, hSUFU and zSUFU did not have the same conformation in solution. Drosophila SUFU is globular and human and zebrafish SUFU are long and dimeric. The N-terminal region involved in hSUFU has been removed and hSUFUΛ30 is present in different oligomerization forms.

No description available.

Les récepteurs à dépendance (RDs) ont la particularité d’induire deux types de signalisation selon la disponibilité de leur ligand. En présence de leur ligand, ils induisent une signalisation positive favorisant la survie, la prolifération ou encore la différentiation cellulaire, tandis qu’en son absence, ils induisent la mort cellulaire par apoptose. Ptc et CDON, tous deux récepteurs du morphogène Shh, appartiennent à la famille des RDs. Ces deux récepteurs sont impliqués dans le développement embryonnaire, mais aussi dans le contrôle de la tumorigenèse et de l’homéostasie. La plupart des mécanismes moléculaires de la signalisation proapoptotique de ces récepteurs sont encore inconnus. Nous avons donc étudié les voies de signalisations proapoptotiques de ces récepteurs grâce à un crible d’ARN interférents. Parmi les partenaires potentiels identifiés, nous nous sommes intéressés à l’IGFBP7, caractérisé comme gène suppresseur de tumeurs dans de nombreux cancers. Nous avons mis en évidence que la mort induite par Ptc implique un contrôle transcriptionnel d’IGFBP7 de façon p53 dépendante. Dans un second temps, nous nous sommes intéressés au rôle des voies apoptotiques des récepteurs Ptc et CDON dans des cancers colorectaux associés à des maladies inflammatoires chroniques de l’intestin, pour lesquels il a été montré que le ligand Shh est surexprimé. La surexpression du ligand est l’un des mécanismes d’échappement au contrôle apoptotique exercé par les RDs acquit par les cellules tumorales. Notre étude vise à démontrer que le ciblage de Shh dans ces cancers pourrait induire une réactivation des voies apoptotiques des RDs Ptc et CDON, et ainsi constituer une nouvelle stratégie anti-tumorale / Dependence receptors (DRs) are able to inducing two types of signaling according to their ligand availability. In the presence of their ligand, they induce a positive signaling promoting survival, proliferation or cell differentiation, whereas in its absence, they induce cell death by apoptosis. Ptc and CDON, both receptors of the morphogen Shh, belong to the DRs family. Both receptors are involved in embryonic development, but also in the control of tumorigenesis and homeostasis. Most of the molecular mechanisms of the pro-apoptotic signaling of these receptors remain unknown. We therefore studied both receptors pro-apoptotic pathways through a shRNA screen. Among the identified potential partners, we have been interested in IGFBP7 shown as a tumor suppressor gene in many cancers. Secondly, we studied the role of Ptc and CDON pro-apoptotic pathways in colorectal cancers associated with chronic inflammatory bowel diseases, in which it has been shown that Shh is overexpressed. Overexpression of the ligand is one mechanism of pro-apoptotic control escapement by DRs acquired by tumor cells. Our study aims to demonstrate that targeting Shh in these cancers could induce reactivation of DRs pro-apoptotic pathways and thus constitute a new anti-tumor strategy