novel insights into the genetics and pathophysiology of adrenocortical tumors

International audienceAdrenocortical tumors (ACTs) are typically unilateral and can be classified as benign adrenocortical adenomas (ACAs) or malignant adrenocortical cancers (ACCs). In rare cases, tumors may occur in both adrenal glands as micronodular hyperplasia (primary pigmented nodular adrenal dysplasia) or as macronodular hyperplasia (primary bilateral macronodular adrenal hyperplasia, PBMAH). The study of certain tumor predisposition syndromes has improved our understanding of sporadic ACTs. Most ACAs are associated with abnormalities of the cAMP signaling pathway, whereas most ACCs are linked to alterations in IGF2, TP53, or the Wnt/βcatenin pathways. Over the past year, single-nucleotide polymorphism array technology and next-generation sequencing have identified novel genetic alterations in ACTs that shed new light on the molecular mechanisms of oncogenesis. Among these are somatic mutations of PKA catalytic subunit alpha gene (PRKACA) in ACA, germline, and somatic mutations of armadillo repeat containing 5 gene (ARMC5) in primary bilateral macronodular adrenal hyperplasia and somatic alterations of the E3 ubiquitin ligase gene ZNRF3 in ACC. This review focuses on the recent discoveries and their diagnostic, prognostic, and therapeutic implications

Combined transcriptome studies identify AFF3 as a mediator of the oncogenic effects of β-catenin in adrenocortical carcinoma

International audienceAdrenocortical cancer (ACC) is a very aggressive tumor, and genomics studies demonstrate that the most frequent alterations of driver genes in these cancers activate the Wnt/β-catenin signaling pathway. However, the adrenal-specific targets of oncogenic β-catenin-mediating tumorigenesis have not being established. A combined transcriptomic analysis from two series of human tumors and the human ACC cell line H295R harboring a spontaneous β-catenin activating mutation was done to identify the Wnt/β-catenin targets. Seven genes were consistently identified in the three studies. Among these genes, we found that AFF3 mediates the oncogenic effects of β-catenin in ACC. The Wnt response element site located at nucleotide position − 1408 of the AFF3 transcriptional start sites (TSS) mediates the regulation by the Wnt/β-catenin signaling pathway. AFF3 silencing decreases cell proliferation and increases apoptosis in the ACC cell line H295R. AFF3 is located in nuclear speckles, which play an important role in RNA splicing. AFF3 overexpression in adrenocortical cells interferes with the organization and/or biogenesis of these nuclear speckles and alters the distribution of CDK9 and cyclin T1 such that they accumulate at the sites of AFF3/speckles. We demonstrate that AFF3 is a new target of Wnt/β-catenin pathway involved in ACC, acting on transcription and RNA splicing

Combined transcriptome studies identify AFF3 as a mediator of the oncogenic effects of beta-catenin in adrenocortical carcinoma

Adrenocortical cancer (ACC) is a very aggressive tumor, and genomics studies demonstrate that the most frequent alterations of driver genes in these cancers activate the Wnt/beta-catenin signaling pathway. However, the adrenal-specific targets of oncogenic beta-catenin-mediating tumorigenesis have not being established. A combined transcriptomic analysis from two series of human tumors and the human ACC cell line H295R harboring a spontaneous beta-catenin activating mutation was done to identify the Wnt/beta-catenin targets. Seven genes were consistently identified in the three studies. Among these genes, we found that AFF3 mediates the oncogenic effects of beta-catenin in ACC. The Wnt response element site located at nucleotide position - 1408 of the AFF3 transcriptional start sites (TSS) mediates the regulation by the Wnt/beta-catenin signaling pathway. AFF3 silencing decreases cell proliferation and increases apoptosis in the ACC cell line H295R. AFF3 is located in nuclear speckles, which play an important role in RNA splicing. AFF3 overexpression in adrenocortical cells interferes with the organization and/or biogenesis of these nuclear speckles and alters the distribution of CDK9 and cyclin T1 such that they accumulate at the sites of AFF3/speckles. We demonstrate that AFF3 is a new target of Wnt/beta-catenin pathway involved in ACC, acting on transcription and RNA splicing

O-GlcNAcase:promiscuous hexosaminidase or key regulator of O-GlcNAc signalling?

O-GlcNAc signaling is regulated by an opposing pair of enzymes: O-GlcNAc transferase installs and O-GlcNAcase (OGA) removes the modification from proteins. The dynamics and regulation of this process are only beginning to be understood as the physiological functions of both enzymes are being probed using genetic and pharmacological approaches. This minireview charts the discovery and functional and structural analysis of OGA and summarizes the insights gained from recent studies using OGA inhibition, gene knock-out, and overexpression. We identify several areas of “known unknowns” that would benefit from future research, such as the enigmatic C-terminal domain of OGA

Combined transcriptome studies identify AFF3 as a mediator of the oncogenic effects of beta-catenin in adrenocortical carcinoma

Adrenocortical cancer (ACC) is a very aggressive tumor, and genomics studies demonstrate that the most frequent alterations of driver genes in these cancers activate the Wnt/beta-catenin signaling pathway. However, the adrenal-specific targets of oncogenic beta-catenin-mediating tumorigenesis have not being established. A combined transcriptomic analysis from two series of human tumors and the human ACC cell line H295R harboring a spontaneous beta-catenin activating mutation was done to identify the Wnt/beta-catenin targets. Seven genes were consistently identified in the three studies. Among these genes, we found that AFF3 mediates the oncogenic effects of beta-catenin in ACC. The Wnt response element site located at nucleotide position - 1408 of the AFF3 transcriptional start sites (TSS) mediates the regulation by the Wnt/beta-catenin signaling pathway. AFF3 silencing decreases cell proliferation and increases apoptosis in the ACC cell line H295R. AFF3 is located in nuclear speckles, which play an important role in RNA splicing. AFF3 overexpression in adrenocortical cells interferes with the organization and/or biogenesis of these nuclear speckles and alters the distribution of CDK9 and cyclin T1 such that they accumulate at the sites of AFF3/speckles. We demonstrate that AFF3 is a new target of Wnt/beta-catenin pathway involved in ACC, acting on transcription and RNA splicing

Hyperglycaemic conditions perturb mouse oocyte in vitro developmental competence via beta-O-linked glycosylation of Heat shock protein 90

STUDY QUESTION What is the effect of beta-O-linked glycosylation (O-GlcNAcylation) on specific proteins in the cumulus-oocyte complex (COC) under hyperglycaemic conditions? SUMMARY ANSWER Heat shock protein 90 (HSP90) was identified and confirmed as being O-GlcNAcylated in mouse COCs under hyperglycaemic conditions (modelled using glucosamine), causing detrimental outcomes for embryo development. WHAT IS KNOWN ALREADY O-GlcNAcylation of proteins occurs as a result of increased activity of the hexosamine biosynthesis pathway, which provides substrates for cumulus matrix production during COC maturation, and also for O-GlcNAcylation. COCs matured under hyperglycaemic conditions have decreased developmental competence, mediated at least in part through the mechanism of increased O-GlcNAcylation. STUDY DESIGN, SIZE, DURATION This study was designed to examine the effect of hyperglycaemic conditions (using the hyperglycaemic mimetic, glucosamine) on O-GlcNAc levels in the mouse COC, and furthermore to identify potential candidate proteins which are targets of this modification, and their roles in oocyte maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS COCs from 21-day-old superovulated CBA × C57BL6 F1 hybrid female mice were matured in vitro (IVM). Levels of O-GlcNAcylated proteins, HSP90 and O-GlcNAc transferase (OGT, the enzyme responsible for O-GlcNAcylation) in COCs were measured using western blot, and localization observed using immunocytochemistry. For glycosylated HSP90 levels, and to test OGT-HSP90 interaction, immunoprecipitation was performed prior to western blotting. Embryo development was assessed using in vitro fertilization and embryo culture post-maturation. MAIN RESULTS AND THE ROLE OF CHANCE Addition of the hyperglycaemic mimetic glucosamine to IVM medium for mouse COCs increased detectable O-GlcNAcylated protein levels (by western blot and immunocytochemistry), and this effect was reversed using an OGT inhibitor (P < 0.05). HSP90 was identified as a target of O-GlcNAcylation in the COC, and inhibition of HSP90 during IVM reversed glucosamine-induced decreases in oocyte developmental competence (P < 0.05). We also demonstrated the novel finding of an association between HSP90 and OGT in COCs, suggesting a possible client–chaperone relationship. LIMITATIONS, REASONS FOR CAUTION In vitro maturation of COCs was used so that treatment time could be limited to the 17 h of maturation prior to ovulation. Additionally, glucosamine, a hyperglycaemic mimetic, was used because it specifically activates the hexosamine pathway which provides the O-GlcNAc moieties. The results in this study should be confirmed using in vivo models of hyperglycaemia and different HSP90 inhibitors. WIDER IMPLICATIONS OF THE FINDINGS This study leads to a new understanding of how diabetes influences oocyte competence and provides insight into possible therapeutic interventions based on inhibiting HSP90 to improve oocyte quality.L.A. Frank, M.L. Sutton-McDowall, H.M. Brown, D.L. Russell, R.B. Gilchrist, and J.G. Thompso

Characterization of O-GlcNAc cycling and proteomic identification of differentially O-GlcNAcylated proteins during G1/S transition in human epithelial cells

La O-GlcNAcylation est une glycosylation dynamique et réversible sous le contrôle de la O-GlcNAc Transférase (OGT) qui transfère un résidu de GlcNAc sur les Ser/Thr de protéines intracellulaires, et de la O-GlcNAcase (OGA). Plusieurs travaux dont ceux de notre équipe ont montré l'importance de la dynamique de O-GlcNAcylation pour la progression normale du cycle cellulaire, et plus particulièrement de la mitose. L’objectif de mes travaux de thèse était de comprendre comment la balance O-GlcNAc participe au contrôle des étapes précoces du cycle cellulaire. J’ai d’abord montré dans différentes lignées cellulaires que l’entrée en phase S s’accompagne d’une baisse marquée du niveau de O-GlcNAc, corrélée à une augmentation de l’expression et de l’activité de l’OGA endogène. Par protéomique, 58 protéines cytosoliques et nucléaires différentiellement O-GlcNAcylées à la transition G1/S ont ensuite été identifiées dans les cellules MCF7 synchronisées. Ces protéines interviennent dans des processus cellulaires essentiels à la phase G1 dont la régulation de la transcription, de la traduction et de la mise en conformation des protéines, et de la réplication de l’ADN. Par immunoprécipitation, les variations O-GlcNAc dépendantes du cycle cellulaire ont été confirmées sur les protéines cytosoliques CK8, hnRNP K et Caprine 1, et sur les protéines nucléaires du complexe de pré-réplication, MCM-3, -4, -6, et -7. Ces travaux montrent donc que la transition G1/S est étroitement liée à la dynamique de O-GlcNAcylation et soulignent un rôle potentiel de cette glycosylation dans le contrôle de l’initiation de la réplication de l’ADN et par là même, dans le maintien de l'intégrité génomique.O-GlcNAcylation is a highly dynamic and reversible glycosylation which is governed by O-GlcNAc Transferase (OGT) that transfers the N-acetylglucosamine (GlcNAc) residue onto Ser/Thr of intracellular proteins, and O-GlcNAcase (OGA). Over the last decade, we and others have shown that dynamics of O-GlcNAcylation was important in regulating the cell cycle progression, and more particularly the mitosis events. The aim of my work was to explore how O-GlcNAc balance is implicated in the control of cellular proliferation by focusing on the early steps in the cell cycle. We highlighted in several cell lines that S-phase entry is associated with a marked decrease in the overall level of O-GlcNAcylated proteins, concordant with an increase in both the expression and activity of endogenous OGA. Then, using a proteomic approach we identified 58 cytoplasmic and nuclear proteins differentially O-GlcNAcylated between G0, G1 and S phases. These proteins are involved in key cellular functions that are essential for G1 and S progression, such as protein folding and translation, transcription or DNA replication. By immunoprecipitation, we further confirmed the cell cycle-dependent O-GlcNAc variations of CK8, hnRNP K, Caprin-1, and MCM -3, -4, -6, and -7 proteins which are part of the pre-replicative complex. To conclude, this study shows that there is a close link between the dynamics of O-GlcNAc and G1/S transition and provides a descriptive overview of differentially O-GlcNAcylated proteins at the G1/S transition, highlighting a potential role of O-GlcNAcylation in the initiation of DNA synthesis and therefore, in the maintenance of genome integrity

Etude de la dynamique de O-GlcNAcylation et identification de protéines différentiellement O-GlcNAcylées au cours de la transition G1/S du cycle cellulaire de cellules épithéliales humaines

La O-GlcNAcylation est une glycosylation dynamique et réversible sous le contrôle de la O-GlcNAc Transférase (OGT) qui transfère un résidu de GlcNAc sur les Ser/Thr de protéines intracellulaires, et de la O-GlcNAcase (OGA). Plusieurs travaux dont ceux de notre équipe ont montré l'importance de la dynamique de O-GlcNAcylation pour la progression normale du cycle cellulaire, et plus particulièrement de la mitose. L objectif de mes travaux de thèse était de comprendre comment la balance O-GlcNAc participe au contrôle des étapes précoces du cycle cellulaire. J ai d abord montré dans différentes lignées cellulaires que l entrée en phase S s accompagne d une baisse marquée du niveau de O-GlcNAc, corrélée à une augmentation de l expression et de l activité de l OGA endogène. Par protéomique, 58 protéines cytosoliques et nucléaires différentiellement O-GlcNAcylées à la transition G1/S ont ensuite été identifiées dans les cellules MCF7 synchronisées. Ces protéines interviennent dans des processus cellulaires essentiels à la phase G1 dont la régulation de la transcription, de la traduction et de la mise en conformation des protéines, et de la réplication de l ADN. Par immunoprécipitation, les variations O-GlcNAc dépendantes du cycle cellulaire ont été confirmées sur les protéines cytosoliques CK8, hnRNP K et Caprine 1, et sur les protéines nucléaires du complexe de pré-réplication, MCM-3, -4, -6, et -7. Ces travaux montrent donc que la transition G1/S est étroitement liée à la dynamique de O-GlcNAcylation et soulignent un rôle potentiel de cette glycosylation dans le contrôle de l initiation de la réplication de l ADN et par là même, dans le maintien de l'intégrité génomique.O-GlcNAcylation is a highly dynamic and reversible glycosylation which is governed by O-GlcNAc Transferase (OGT) that transfers the N-acetylglucosamine (GlcNAc) residue onto Ser/Thr of intracellular proteins, and O-GlcNAcase (OGA). Over the last decade, we and others have shown that dynamics of O-GlcNAcylation was important in regulating the cell cycle progression, and more particularly the mitosis events. The aim of my work was to explore how O-GlcNAc balance is implicated in the control of cellular proliferation by focusing on the early steps in the cell cycle. We highlighted in several cell lines that S-phase entry is associated with a marked decrease in the overall level of O-GlcNAcylated proteins, concordant with an increase in both the expression and activity of endogenous OGA. Then, using a proteomic approach we identified 58 cytoplasmic and nuclear proteins differentially O-GlcNAcylated between G0, G1 and S phases. These proteins are involved in key cellular functions that are essential for G1 and S progression, such as protein folding and translation, transcription or DNA replication. By immunoprecipitation, we further confirmed the cell cycle-dependent O-GlcNAc variations of CK8, hnRNP K, Caprin-1, and MCM -3, -4, -6, and -7 proteins which are part of the pre-replicative complex. To conclude, this study shows that there is a close link between the dynamics of O-GlcNAc and G1/S transition and provides a descriptive overview of differentially O-GlcNAcylated proteins at the G1/S transition, highlighting a potential role of O-GlcNAcylation in the initiation of DNA synthesis and therefore, in the maintenance of genome integrity.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF