Yes / Sonic Hedgehog (SHH) is a glycoprotein associated with development that is also expressed in the adult CNS and released after brain injury. Since the SHH receptors PTCH1 (patched homolog-1) and SMO (Smoothened) are highly expressed on astrocytes, we hypothesised that SHH regulates astrocyte function. Primary mouse cortical astrocytes derived from embryonic (E15) Swiss mouse cortices, were treated with two chemically distinct agonists of the SHH pathway, which caused astrocytes to elongate and proliferate. These changes are accompanied by decreases in the major astrocyte glutamate transporter, GLT-1 and the astrocyte intermediate filament protein GFAP. Multi-site electrophysiological recordings revealed that the SHH agonist, SAG supressed neuronal firing in astrocyte-neuron co-cultures and this was abolished by the astrocyte metabolic inhibitor ethylfluoroacetate, revealing that SHH stimulation of metabolically-active astrocytes influences neuronal firing. Using 3D co-culture, MAP2 western blotting and immunohistochemistry, we show that SHH-stimulated astrocytes protect neurons from kainate induced cell death. Altogether the results show that SHH regulation of astrocyte function represents an endogenous neuroprotective mechanism. / BBSRC

La forme la plus agressive des gliomes, tumeurs cérébrales primitives, est l'astrocytome de grade IV appelé aussi glioblastome (GBM). Le pronostic des patients atteints d'un GBM est très sombre du fait de la nature infiltrante de ces cancers et de la présence de cellules souches cancéreuses (CSC) peu sensibles aux thérapies actuelles. Il est donc primordial d'étudier ces deux caractéristiques des GBM. Mes travaux de thèse ont porté sur ces deux aspects.La connaissance de l'implication de la voie de signalisation Hedgehog (Hh) dans le maintien du pool de CSC permet d'envisager de nouvelles stratégies thérapeutiques visant à les éliminer. La voie Hh joue un rôle important dans le développement embryonnaire. L'activation de cette voie a été mise en évidence dans de nombreux cancers dont les GBM. La cyclopamine est une substance alcaloïde inhibitrice de la voie Hh. L'inconvénient de l'utilisation de la cyclopamine pour une thérapie anticancéreuse est que cette voie est aussi active dans les cellules souches somatiques adultes. Pour cibler la tumeur, une prodrogue glucuronylée de la cyclopamine a été synthétisée. La partie glucuronide peut être hydrolysée par la β-glucuronidase, une enzyme présente dans la zone nécrotique des GBM, conduisant à la libération de la cyclopamine active de façon ciblée, dans le microenvironnement tumoral. Des tests de viabilité indiquent que cette prodrogue ne présente pas latoxicité de la cyclopamine envers les cellules C6 de GBM de rat, leur population de cellule souches (C6-GSC) ainsi que dans des coupes de cerveau de rat adulte. En revanche, l'hydrolyse par la β-glucuronidase de cette prodrogue permet de restaurer la toxicité de la cyclopamine sur les cellules de GBM. L'analyse par RT-qPCR de l'expression d'un gène cible de la voie Hh, le facteur de transcription GLI1, permet de vérifier l'inhibition spécifique de la voie Hh par la cyclopamine dans les cellules C6 et les C6-GSC. Ainsi, la prodrogue en libérant la cyclopamine dans l'environnement tumoral devrait permettre de limiter l'étendue des effets de la drogue sur les cellules saines du cerveau.La voie Hh joue un rôle dans la régulation de la migration et de l'invasion des cellules de GBM. Mon équipe d'accueil a montré que le vasoactive intestinal peptide (VIP) régule la migration et l'invasion de deux lignées cellulaires M059K et M059J. Dans ce travail de thèse, les effets du VIP et de son analogue le pituitary adenylate cyclase-activating peptide(PACAP) sur la migration et l'invasion des cellules C6 de rat et U87 humaines ont été étudiés. Les résultats obtenus indiquent que les agonistes VIP, PACAP-38 et l'antagoniste synthétique VIP10-28 des récepteurs de ces peptides contrôlent la migration et l'invasion dans les deux lignées de GBM. Le VIP et le PACAP-38 inhibent l'invasion ex vivodes cellules C6 dans des coupes de cerveau. De plus, le VIP10-28 augmente la migration et l'invasion de ces cellules de façon PKA-, Akt- et Hh-dépendante. L'étude des voies detransduction impliquées dans l'action des neuropeptides sur la migration et l'invasion des GBM a ensuite été approfondie. Les données obtenues indiquent que le VIP et le PACAP diminuent le taux de la forme active nucléaire de la protéine GLI1 dans les cellules C6 et U87 de façon PKA-dépendante. Inversement, le VIP10-28 augmente l'expression de GLI1 dans les cellules C6 de façon Akt-dépendante. Enfin, nous avons constaté que le PACAP diminue la phosphorylation d'Akt dans les cellules C6 etaugmente celle de la protéine PTEN, en accord avec le rôle de ces kinases dans la régulation de la migration cellulaire et de l'activité GLI1. L'ensemble de ces résultats indique que le système VIP-récepteurs régule la migration et l'invasion par des interactions entre les voies PKA, Hh et Akt/PTEN dans les cellules de GBM. / The most aggressive form of glioma, primary brain tumors, is grade IV astrocytoma also called glioblastoma (GBM)...

Many signaling pathways which are classically understood for their roles in early development are also known to be involved in tissue maintenance and adult energy homeostasis. Furthermore, dysfunction of these signaling pathways results in human diseases such as cancer. An in depth understanding of how developmentally important signaling pathways function in the adult will provide mechanistic insights into disease and potential new therapeutic targets. Herein Chapter 1, the Wnt, fibroblast growth factor (FGF), and Hedgehog (Hh) signaling pathways are discussed and examples of their relevance in development, adult homeostasis, and disease are provided. Wnt signaling provides an example of this concept as it has well described roles during both development and adult metabolism.<div><br></div><div> Work included in Chapter 2, investigates the regulation of adult energy homeostasis by a member of the endocrine FGF family, FGF19. The three endocrine FGFs, FGF19 (FGF15 in mice), FGF21, and FGF23 have well described roles in the regulation of metabolic processes in adults. While FGF23 is primarily involved in the regulation of phosphate and vitamin D homeostasis,FGF19 and FGF21 have shown similar pharmacological effects on whole body metabolism. Here, the importance of adaptive thermogenesis for the pharmacological action of FGF19 is explored. UsingUCP1KO animals we show that whole-body thermogenesis is dispensable for body weight loss following FGF19 treatment.<br></div><div><br></div><div>Finally, the potential involvement of Hh signaling in mediating the hyperphagia driven obesity observed in certain ciliopathies is explored in Chapter 3. Emerging evidence suggests cilia play an important role in the regulation of feeding behavior. In mammals, the hedgehog pathway is dependent on the primary cilium as an organizing center and defects in hedgehog signaling share some clinical symptoms of ciliopathies. Here, we characterized the expression of core pathway components in the adult hypothalamus. We show that neurons within specific nuclei important for regulation of feeding behavior express Hh ligand and members of its signaling pathway. We also demonstrate that the Hh pathway is transcriptionally upregulated in response to an overnight fast. This work provides an important foundation for understanding the functional role of Hh signaling in regulation of energy homeostasis. In its entirety, this work highlights the emerging clinical relevance of developmentally critical pathways in diseases associated with dysfunction of adult tissue homeostasis, such as obesity.<br></div>

No description available.

Introduction. The inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), are common in Western Europe (200-400 cases /100,000) and associated with substantial morbidity, although mortality is now low. There is presently a great unmet need for novel therapeutics in IBD as present agents are limited by lack of efficacy, toxicity and poor patient acceptance. Recent findings from genome-wide association studies (GWAS) have characterised the genetic architecture of CD and UC. Defects in innate and adaptive immunity have been clearly established, and substantial novel insights into disease pathogenesis have been gained. Over 30 genes / loci are now associated with CD; a number of these, along with a few specific loci, are also associated with UC. The hedgehog (HH) signalling pathway is critical to gastrointestinal development and plays key roles in intestinal and immune homeostasis. Furthermore, in addition to well described roles in tumorigenesis, it is evident that recapitulation of embryonic HH signals play critical roles in response to acute and chronic inflammatory challenge in diverse tissues. Aims. The main aims of the work presented in this thesis were to characterise the expression of key HH signalling components in the healthy and inflamed human intestine, establish whether germline variation in HH genes is associated with IBD and describe the in vitro responses of intestinal epithelial cells to pathogen associated molecular patterns. The WNT pathway, antagonised by HH in the intestine, and two HH target genes (NKX2.3 and CCL20) were also analysed for evidence of association with IBD. Methods. Expression of HH and WNT signalling components was described by immunohistochemistry and microarray analysis in healthy controls (HC), CD, UC, and non- IBD inflamed terminal ileal and colonic samples. Gene-wide haplotype-tagging studies were performed for GLI1 in Scottish, English and Swedish CD and UC, and Scottish early-onset colo-rectal cancer, IHH in Scottish IBD, NKX2.3 in Scottish and UK IBD, and CCL20 in Scottish, Swedish and Japanese IBD. Evidence for association of all HH (n=13) and WNT (n=27) signalling genes in CD was established by analysis of UK GWAS data and metaanalysis from UK, French/Belgium and N American studies. The effect of lipopolysaccharide (LPS) and muramyl dipeptide (MDP) on HH signalling was assessed in colonic epithelial cells (SW480). The effect of HH pathway agonists and antagonists on NFκB activity and cytokine expression was analysed in SW480 cells and peripheral blood mononuclear cells (HC and IBD patients) in vitro. Results. The expression of HH pathway ligand is present in the intestinal epithelium and the pathway response network in the lamina propria demonstrating the paracrine nature of HH signalling in the intestine. Immunohistochemical studies and microarray analysis demonstrates that HH pathway activity is decreased in all forms of colonic inflammation studied in man. Variation in Glioma-associated oncogene homolog 1 (GLI1), a key HH transcription factor located at 12q13 (IBD2), was associated with IBD (p<0.0001), UC (p<0.0001) and to a lesser extent CD (p=0.03) in Scotland, a finding replicated in English IBD and UC. This association was attributed to a non-synonymous SNP (rs2228226C→G) with pools odds ratio of 1.194 in meta-analysis of over 5000 individuals from Scotland, England and Sweden (p=0.0002). There was association of this SNP with early-onset colorectal cancer, but of borderline significance (p=0.05). The variant protein (Q1100E) is 50% less active than wild-type protein in vitro. IHH was not associated with CD or UC. Preliminary evidence was produced for association at SUFU (10q24; p=0.005), a GLI1- binding protein, and at the WNT3 / WNT9B locus (17q21; p=0.0005). MDP stimulation of colonic epithelial cells decreased HH pathway activity. Exogenous HH increased expression of CCL20. CCL20 promoter polymorphisms were associated with UC in Japanese patients (p=0.018) but not in Scotland or Sweden. NKX2.3 was associated with IBD in Scotland (UC>CD), but there was insufficient power for fine-mapping of causative variants. Conclusions. Multiple lines of evidence presented here demonstrate that the HH signalling pathway is involved in IBD pathogenesis. In key complementary in vivo studies (conceived by CWL; conducted in collaboration with the Gumucio lab in Ann Arbor) we have demonstrated that Gli1+/- mice develop early, severe colitis with high mortality in response to acute inflammatory challenge. Furthermore, lamina propria antigen presenting cells are identified as the key HH target cells. With HH agonists and antagonists in extensive preclinical and early clinical testing, these studies have real potential to translate into novel therapeutics for patients with IBD.

Με την εργασία έγινε μελέτη του σηματοδοτικού μονοπατιού Sonic Hedgehog σε 96 περιστατικά καρκίνου πνεύμονα με τη μέθοδο της ανοσοϊστοχημείας. Επίσης μελετήσαμε την πιθανή συμμετοχή του μεταγραφικού παράγοντα FoxM1 στο καρκίνωμα του πνεύμονα και την πιθανή συσχέτισή του με το μονπάτι του Hedgehog. Έγινε μελέτη της έκφρασης των μορίων Shh, Ptch1, Smo, Gli1, Gli2 και FoxM1. Τα αποτελέσματα αποκάλυψαν μια έντονη έκφραση των μορίων του μονοπατιού και αυξημένα ποσοστά ενεργοποίησής του. Επίσης βρέθηκε στατιστικά σημαντική συσχέτιση με τα πλακώδη καρκινώματα και με τα χαμηλού grade καρκινώματα. Ανάλογη σημαντική συσχέτιση βρέθηκε και με το φύλο,συχνότερα ενεργοποιημένο μονοπάτι στους άντρες. Ακόμα ανιχνεύτηκε μια συσχέτιση της έκφρασης του FoxM1 με το ενεργοποιημένο μονοπάτι. / The hedgehog (HH)-signaling pathway is implicated in developmental processes and its aberrant activation in adult tissues has been associated with malignancy. The aim of this study was to determine the expression pattern of HH-signaling molecules in lung carcinomas, as well as the involvement of the transcription factor FOXM1, that controls cell proliferation, in this process. Paraffin-embedded tissue sections of 96 lung cancer cases and adjacent non-neoplastic lung parenchyma were immunohistochemically analyzed with anti-SHH, anti-Patched1 (PTCH1), anti-Smoothened (SMO), anti-GLI1, anti-GLI2 and anti-FOXM1 antibodies. Correlations of HH molecules with clinicopathological parameters and FOXM1 expression were evaluated. All the HH-signaling molecules examined were overexpressed in lung cancer compared with the adjacent non-neoplastic lung parenchyma. HH pathway activity and expression of PTCH1 and SMO were significantly higher in squamous cell carcinomas compared to other histological types. Activation of HH pathway and PTCH1 expression were correlated with tumor grade being higher in low grade tumors. There was a significant correlation of lymph node metastases with expression of SMO in all histological types and with the gender higher in men. Overexpression of FOXM1 in lung cancer was also significantly correlated with PTCH1, SMO and GLI1 expression. In conclusion, HH-signaling pathway is activated in lung cancer and correlates with histological type, prognostic parameters of the tumors as well as with the increased expression of FOXM1.

No description available.

La métastase et la chimiorésistance sont les principales causes de mortalité chez les patients atteints d’un cancer. La compréhension des mécanismes moléculaires régissant ces deux processus devient donc un domaine de recherche important pour la conception de nouvelles stratégies thérapeutiques. Dans ma thèse, je me concentre sur la compréhension du rôle du facteur d’initiation de la traduction chez les eucaryotes 4E (eIF4E) dans l’invasion du cancer, et je décris un nouveau mécanisme de résistance que nous avons découvert en étudiant le développement de la résistance à un inhibiteur connu d’eIF4E, la ribavirine. eIF4E est un puissant oncogène qui est connu pour être élevé dans une multitude de cancers comprenant entre autres les sous-types M4 / M5 de la leucémie myéloïde aiguë (AML). Il fonctionne dans la traduction et l'exportation nucléocytoplasmique d'ARNm en se liant à la coiffe m7G des ARNm possédant des codes USER spécifiques dans leur région UTR 5' et/ou 3'. En reconnaissant ces codes USER, le complexe dans lequel se trouve eIF4E régule de manière coordonnée l'expression de gènes essentiels à la croissance, à la prolifération et à la survie, et ainsi, eIF4E a été placée en tant que nœud central d'un régulon d'ARN régissant la prolifération. En analysant les voies dans lesquelles l’export est régulé de façon coordonnée par eIF4E et les effets physiologiques qui en découlent, j'ai trouvé un enrichissement de la voie biosynthétique de l'acide hyaluronique (HA) et de son principal récepteur CD44 qui sont des médiateurs clés connus des métastases cancéreuses. J’ai également démontré que l'élévation d’eIF4E modifie la surface des cellules cancéreuses en les recouvrant de protrusions riches en HA de type microvillus et enrichies d'armes de destruction métastatique. Heureusement, en dégradant le manteau HA ou en utilisant des inhibiteurs de CD44 en combinaison avec la ribavirine, nous pouvons alors nous défendre. Compte tenu de l'avantage prolifératif que confère la surexpression d’eIF4E, il est devenu un talon d'Achille attrayant pour le traitement de cancers ayant un niveau élevé d'eIF4E. En effet, lors d'un essai clinique de phase II parmi des patients atteints de leucémie myéloïde aiguë M4 / M5 réfractaire et récidivante, la ribavirine a conduit au ciblage d'eIF4E et a donné lieu à des réponses cliniques significatives, incluant des réponses complètes ou partielles. Cependant, tel qu’attendu lors d’un traitement monothérapique, les patients ayant répondu finissent par développer une résistance au médicament. Mon analyse a révélé que cette résistance est due à un mécanisme nouveau caractérisé par l'élévation du facteur de transcription Sonic Hedgehog GLI1 qui conduit à la glucuronidation du médicament et donc à la perte de l'interaction entre la drogue et sa cible. Heureusement, ce mécanisme peut être inversé en utilisant des inhibiteurs de la voie Hedgehog. En conclusion, ces découvertes fournissent de nouvelles cibles thérapeutiques pour le traitement des cellules cancéreuses agressives et résistantes. / Metastasis and chemoresistance are the leading cause of mortality among cancer patients. The discovery of molecular mechanisms governing these two processes is becoming an important area of research for the design of novel therapeutic strategies. In my thesis, I focus on understanding the role of the eukaryotic translation initiation factor 4E (eIF4E) in cancer invasion and describe a novel mechanism of resistance that we discovered while studying the development of resistance to a known eIF4E inhibitor, ribavirin. eIF4E is a potent oncogene that is known to be elevated in a multitude of cancers including M4/M5 subtypes of acute myeloid leukemia (AML). It functions in mRNA translation and nucleocytoplasmic export by binding to the m7G cap of mRNAs possessing specific USER codes in their 5’ and/or 3’ UTRs. By recognizing these USER codes, eIF4E complex coordinately regulates the expression of genes essential for growth, proliferation and survival and as such has been placed as a central node of an RNA regulon governing proliferation. When analyzing which pathways have their export coordinately regulated by eIF4E and what physiological effects arise from it, I found an enrichment in the hyaluronic acid (HA) biosynthetic pathway as well as its major receptor CD44 which are known key mediators of cancer metastasis. I demonstrate that eIF4E elevation changes the surface of cancer cells sugar-coating them with HA-rich microvillus-like protrusions that are enriched with weapons of metastatic destruction. Luckily, through degrading the HA-coat or using inhibitors of CD44 in combination with ribavirin we can strike back. Given the proliferative advantage that eIF4E overexpression conveys, this rendered it as an attractive Achilles heel for the treatment of cancers where eIF4E levels are high. Indeed, in a phase II clinical trial in refractory and relapsed poor prognosis M4/M5 AML patients, ribavirin led to eIF4E targeting and resulted in significant clinical responses including complete and partial remissions. However, as it is expected for monotherapy treatment, all responding patients eventually developed resistance to the drug. My analysis revealed that resistance is due to a novel mechanism characterized by elevation of the Sonic Hedgehog transcription factor GLI1 which leads to drug glucuronidation and the subsequent loss of drug-to-target interaction. Fortunately, this mechanism can be reversed using Hedgehog pathway inhibitors. Taken together, these findings provide novel therapeutic venues for the treatment of aggressive and resistant cancer cells.

No description available.