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No description available.

Opioid receptor-mediated regulation of chemokine receptors is vital for the host immune response, development, and neurological function. Previous studies have demonstrated that the kappa opioid receptor (KOR) activation results in decreased infectivity of human immunodeficiency virus 1 (HIV-1) in human peripheral blood mononuclear cells (PBMCs). We have found this effect is due to down-regulation of the major HIV-1 co-receptors, CCR5 and CXCR4. Using molecular techniques, CCR5 and CXCR4 mRNA levels drop dramatically following KOR activation. To dissect the mechanism involved, we used transcription factor binding arrays and compared control cell extracts to KOR activated cell extracts. We determined that the interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) could be involved in the KOR-mediated repression of CCR5 and CXCR4 transcription and protein expression. Using chemical inhibitors and small interfering RNA (siRNA) molecules, we determined that JAK2, STAT3, and IRF2 are critical members of this signal transduction pathway. The understanding of these particular mechanisms should prove to be beneficial for the development of potential pharmacological agents targeted at HIV-1 binding and infection since virus infection requires expression of the co-receptors CXCR4 and CCR5. Understanding the molecular basis for KOR-induced inhibition of co-receptor expression may provide a basis for the development of KOR agonist-based therapeutics to treat individuals infected with HIV. / Molecular Biology and Genetics

Nescient Helix Loop Helix 2 (Nhlh2) is a member of the basic helix-loop-helix transcription factor family. Mice with a targeted deletion of Nhlh2, called N2KO mice, show adult onset obesity in both males and females. Nhlh2 regulates other genes by binding to the E-box in the promoter region of these genes. This transcription factor regulates many other transcription factors including MC4R and PC1/3 which are associated with human obesity. The Nhlh2 promoter has been analyzed for putative transcription factors binding sites. These putative binding sites have been tested to be the regulators of Nhlh2 by transactivation assays with mutant promoters, Electrophoretic Shift Assay (EMSA), and Chromatin Immunoprecipitation Assay (ChIP) as methods to investigate the DNA-protein binding. The results of these experiments showed that the Nhlh2 promoter has five Signal Transducer and Activator of Transcription 3 (Stat3) binding site motifs at -47, -65, -80, -281, -294 and two Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells (NFκB) binding site motifs at -67 and -135. While NFκB acts as a negative regulator of Nhlh2, this research showed that Stat3 acts as a regulator for the Nhlh2 basal expression and leptin stimulation. The ChIP assay using chromatin from mouse hypothalamus and antibodies against Stat3 and the NFκB subunits P50, P65, and c-Rel demonstrated that all of these antibodies were able to pull down the part of the Nhlh2 promoter containing the binding sites of Stat3 and NFκB. The EMSA results not only demonstrated that NFκB and Stat3 binding site motifs are real binding sites, but also exists the possibility of a relationship between these transcription factors to regulate Nhlh2 expression with leptin stimulation. An effort in analyzing the human NHLH2 3'UTR showed that one of the SNPs located at position 1568 in the NHLH2 mRNA (NHLH2A^1568G) which converts adenosine to guanine might have the potential to decrease the mRNA stability. For more investigation about this SNP, the mouse Nhlh2 tail was cloned into 2 different vectors and these vectors were subjected to site directed mutagenesis to create the 3'UTR SNP that convert A to G. One of these vectors used luciferase as a reporter gene for expression while the other one was used to measure Nhlh2 mRNA stability. These vectors were transfected into hypothalamic cell line N29/2 to test the effect of this SNP on Nhlh2 expression. This study demonstrated that this SNP down regulated luciferase expression and also decreased Nhlh2 mRNA stability. Taken together, this study demonstrated that Nhlh2 could be regulated transcriptionally by both NFκB and Stat3 transcription factors and post-transcripitionally by the 3'UTR SNP that converts adenosine to guanine. / Ph. D.

Many human tumors harbor persistently-active Signal Transducer and Activator of Transcription (ST AT)3 protein. There is substantial evidence that aberrantly-active STAT3 is a master regulator of events that promote carcinogenesis and human tumor formation. Abnormal STAT3 activity induces uncontrolled growth and survival of cells, thereby contributing to neoplastic transformation and progression. Cisplatin is a major chemotherapeutic modality for ovarian cancer, but is frequently challenged by drug resistance. Given that STAT3 is aberrantly-active in many human tumors, including ovarian cancer, there is the potential that it contributes to the development of Cisplatin resistance, a problem ripe for investigation. This study was conducted to explore the potential that the aberrant STAT3 present in ovarian cancer cells contributes to the decreased sensitivity to Cisplatin observed for ovarian cancer cells. The investigation revealed that STAT3 is aberrantly activated in cancer cell lines resistant to Cisplatin, but not in sensitive cells. Inhibition of aberrant STAT3 activity by the small-molecule STAT3 inhibitor, NSC 74859, increased growth inhibition induced by Cisplatin in resistant ovarian cancer cells. Furthermore, NSC 74859 enhanced apoptosis induced by Cisplatin in resistant cells in vitro by nearly 52%. Collectively, these observations indicate that inhibition of hyperactive STAT3 increases Cisplatin sensitivity, and therefore effectiveness, in resistant cells. Thus, STAT3 represents a viable target for enhancing the sensitivity of ovarian cancer cells to Cisplatin.

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Les adénomes hépatocellulaires (AHC) sont des tumeurs bénignes rares qui se développent le plus souvent chez la femme jeune suite à la prise de contraceptifs oraux. Les complications principales sont l’hémorragie et plus rarement, la transformation maligne en carcinome hépatocellulaire (CHC). Des travaux récents ont permis d’identifier 3 groupes moléculaires principales d’AHC qui se définissent par (1) l’inactivation du facteur de transcription HNF1A (H-HCA), (2) l'activation de la voie Wnt/ß-caténine (bHCA) ou (3) la présence d’infiltrats inflammatoires (IHCA).Afin d’identifier les voies de tumorigenèse associées au développement d’AHC inflammatoires (IHCA), une analyse transcriptomique comparant des IHCA à des foies non tumoraux a été réalisée au laboratoire, ce qui a permis d’identifier dans ce groupe tumoral une activation de la voie IL-6/JAK/STAT3. Nous avons recherché de nouvelles altérations géniques et nous avons caractérisé le mécanisme d'activation de la voie IL-6/JAK/STAT dans les IHCA. Les conséquences fonctionnelles sur la voie STAT3 des différents mutants ont été analysées par une modélisation de leur expression dans des lignées hépatocellulaires. Par ailleurs, nous avons réalisé des études génomiques intégrées (analyse CGH-SNP, méthylome et séquençage exome) sur une large série de 250 AHC avec pour objectif d’affiner la classification moléculaire des AHC, d’identifier de nouveaux gènes altérés dans ces tumeurs et d’élucider les mécanismes de transformation maligne des AHC en CHC.Dans le groupe des IHCA, ces analyses nous ont permis d’identifier de nombreux oncogènes activés par mutation somatique ; de plus, trois de ces gènes n’avaient jamais été décrits comme étant mutés dans des tumeurs humaines. Nous avons identifié des mutations activatrices du récepteur à l’IL-6, gp130 dans 60% des IHCA. Nous avons aussi retrouvé des mutations de FRK, une src-like kinase, dans 10% des IHCA, du facteur de transcription STAT3 dans 5% des IHCA, du gène GNAS dans 5% des cas, et de la tyrosine kinase JAK1 dans 1% des cas. Toutes les mutations identifiées étaient somatiques, monoalléliques et mutuellement exclusives. Nous avons pu montrer, dans des systèmes de lignées cellulaires hépatocellulaires, que l'expression des formes mutées de ces gènes est capable d’activer la voie IL-6/STAT3 en absence du ligand IL-6, contrairement aux protéines sauvages. Nous avons identifié des inhibiteurs pharmacologiques qui permettent d’inhiber de façon spécifique ces mutants et qui pourraient être utilisés en clinique pour le traitement des IHCA.Grâce à une technique de CGH-SNP, nous avons identifié des événements récurrents de pertes et gains de chromosomes associés aux groupes moléculaires d’AHC. De façon similaire, l’étude de la méthylation dans les AHC a permis de mettre en évidence un pattern spécifique à chaque sous groupe. Nous avons montré que l’instabilité chromosomique augmente progressivement dans les lésions borderline et dans les CHC développés sur AHC comparés aux AHC classiques. Le séquençage exome de 5 transformations malignes de AHC en CHC a identifié un nombre plus important de mutations dans les AHC qui ont transformé comparé aux AHC classiques ; ce nombre est significativement augmenté dans la partie CHC des tumeurs. La comparaison de la partie bénigne et maligne des tumeurs a mis en évidence l'activation de ß-caténine comme un évènement précoce dans le processus de transformation et a révélé la présence de mutations somatiques fréquentes dans le promoteur de la télomèrase (TERT), identifiées principalement dans la partie maligne des tumeurs.En conclusion, cette étude a permis d’identifier des mécanismes distincts conduisant à l'activation de STAT3 dans les IHCA, renforçant le rôle de la voie JAK-STAT3 dans la tumorigenèse bénigne hépatocellulaire ainsi que le lien entre Src kinases et inflammation. Ces travaux ont permis d’affiner la classification moléculaire des AHC avec des corrélations étroites... / Hepatocellular adenomas (HCA) are rare benign tumors that develop most often in young women after taking oral contraception. The main complications are hemorrhage and rarely, malignant transformation to hepatocellular carcinoma (HCC). Recent work in the laboratory identified three main HCA molecular groups that are defined by (1) inactivation of the transcription factor HNF1A (H-HCA), (2) activation of the Wnt/ß-catenin pathway (bHCA) or (3) the presence of inflammatory infiltrates (IHCA).To identify tumorigenesis pathways associated with the development of inflammatory HCA (IHCA), a transcriptome analysis comparing IHCA to non-tumor liver was performed in the laboratory, leading to the identification of an activation of the IL-6/JAK/STAT3 pathway in these tumors. We sought new gene alterations and we characterized the activation mechanism of the IL-6/JAK/STAT pathway in IHCA. The functional consequences of the different mutants on the STAT3 pathway were analyzed by modeling their expression in hepatocellular cell lines. In addition, we performed integrated genomic studies (CGH-SNP analysis, methylome and exome sequencing) on a wide range of 250 HCA with the aim to refine the molecular classification of HCA, to identify new genes altered in these tumors and to elucidate the mechanisms of malignant transformation of HCA to HCC.In the group of the IHCA, we identified many oncogenes activated by somatic mutation; in addition, three of these genes were never been described as mutated in human tumors. We identified activating mutations in the IL-6 receptor gp130 in 60% of IHCA. We also found mutations in FRK, a src-like kinase, in 10% of IHCA, of the transcription factor STAT3 in 5% of IHCA, of the GNAS gene in 5% of cases, and of the tyrosine kinase JAK1 in 1% of the cases. All identified mutations were somatic and monoallelic and were mutually exclusive. We have shown in hepatocellular cell lines that the expression of mutated forms of these genes is able to activate the IL-6/STAT3 pathway in the absence of the IL-6 ligand, in contrast to wild-type proteins. We have identified pharmacological inhibitors that specifically inhibit the mutants and that could be used for the clinical treatment of IHCA.Using a CGH-SNP technique, we identified recurrent chromosomes gains and losses associated with the HCA molecular groups. Similarly, the study of methylation in HCA highlighted a specific pattern in each subgroup. We showed that chromosomal instability increases gradually in borderline lesions and in HCC developed on HCA compared to classical HCA. Exome sequencing of 5 malignant transformation of HCA to HCC identified a large number of mutations in the transformed HCA compared to classical HCA; and this number is significantly increased in HCC tumors counterpart. Comparison of benign and malignant tumors highlighted the activation of ß-catenin as an early event in the transformation process and revealed frequent somatic mutations in the promoter of the telomerase gene (TERT), identified mainly in the malignant part of tumors.In conclusion, this study has led to the identification of distinct mechanisms leading to the activation of STAT3 in IHCA, strengthening the role of the JAK-STAT3 pathway in benign hepatocellular tumorigenesis and the relationship between Src kinases and inflammation. This work helped to refine the molecular classification of HCA with tight correlations between genotype and phenotype, and led to advances in the identification of major genetic determinants involved in the process of malignant transformation.

Chez la souris, le Leukemia Inhibitory Factor (LIF) joue un rôle clé dans le maintien des cellules souches embryonnaires (ES) à l’état pluripotent. Le LIF agit en activant le facteur de transcription STAT3 via les kinases Jak. Cette activation est nécessaire et suffisante au maintien des cellules ES en autorenouvellement en présence de sérum. Une étude du transcriptome de STAT3 réalisée au laboratoire a permis d’identifier plusieurs gènes cibles de ce facteur, parmi lesquels plusieurs gènes inconnus. L’un d’eux, le gène 1456160_at, est fortement exprimé dans les cellules ES de souris et son expression diminue après induction de la différenciation. Ce gène a été appelé Asgard pour Another Self-renewal GuARDian. La caractérisation et le séquençage de ce gène ont permis de mettre en évidence qu'Asgard code pour un microARN. De nombreux microARNs jouent un rôle clé dans le maintien de l'autorenouvellement des cellules ES et dans le contrôle de la différenciation. Des expériences d’inhibition et de surexpression ont permis de montrer que Asgard est impliqué dans la régulation de la différenciation endoderme versus mésoderme. Des analyses préliminaires ont permis d’identifier Pbx3, FoxA2 et Sox17 comme cibles potentielles. Bien que les mécanismes d’action du microARN Asgard restent à confirmer, ce travail a permis d’identifier un nouveau gène clé de l'autorenouvellement des cellules ES de souris / The Leukemia Inhibitory Factor (LIF) activates the transcription factor STAT3, which results in the maintenance of mouse embryonic stem cells in the undifferentiated state by inhibiting mesodermal and endodermal differentiation. We identified several target genes of STAT3 by transcriptomic analysis. Among them, we focused on an unknown gene referred as 1456160_at on Affymetrix array. This gene is highly expressed in embryonic stem cells and its expression level decreases during differentiation. We named this gene Asgard for Another Self-renewal GuARDian. Its characterization and sequencing revealed that Asgard encodes for a microRNA sequence. Several microRNAs have been shown to play key role in the maintenance of self-renewal of mouse ES cells and in the control of differentiation. Inhibition and overexpression assays showed that Asgard inhibits endodermal differentiation in order to maintain self-renewal. Through preliminary analysis, we identified Pbx3, FoxA2 and Sox17 as potential targets of the microRNA Asgard. Our work enables us to identify a new key gene of self-renewal of mouse ES cells