The type II tumour suppressor ING3 has been shown to modulate transcription, cell cycle control, and apoptosis. To investigate the putative role of ING3 in melanoma development, we examined the expression of ING3 in 58 dysplastic nevi, 114 primary melanomas, and 50 metastatic melanomas with tissue microarray and immunohistochemistry. Overall ING3 was reduced in metastatic melanomas compared with dyslastic nevi and primary melanomas. Reduced nuclear ING3 staining also correlated with melanoma progression, increased cytoplasmic ING3 level, tumour location at sun-exposed sites, and a poorer disease-specific 5-year survival of patients with primary melanoma. Multivariate analysis revealed that nuclear ING3 staining can independently predict patient outcome in primary melanomas. In melanoma cells, ING3 expression was rapidly induced by UV irradiation. Using stable clones of melanoma cells overexpressing ING3, we showed that ING3 significantly promoted UV-induced apoptosis. Unlike its homologues ING1b and ING2, ING3-enhanced apoptosis upon UV irradiation was independent of functional p53. Furthermore, ING3 did not affect the expression of mitochondrial proteins but increased the cleavage of Bid and caspases. Moreover, ING3 upregulated Fas expression and ING3-mediated apoptosis was blocked by inhibiting caspase-8 or Fas activation. Knockdown of ING3 expression decreased UV-induced apoptosis remarkably, suggesting that ING3 plays a crucial role in cellular response to UV radiation. To explore how ING3 is deregulated in advanced melanomas, we examined ING3 expression in metastatic melanoma cells and found that ING3 was downregulated due to a rapid protein turnover in these cells. Further studies demonstrated that ING3 undergoes degradation via the ubiquitin-proteasome pathway. We also demonstrate that ING3 interacts with the SCF (Skp1/Cul1/Roc1/Skp2) E3 ligase complex. Knockdown of Cul1 or Skp2 significantly stabilized ING3 in melanoma cells. In addition, lysine residue 96 is essential for ING3 ubiquitination as its mutation to arginine completely abrogated ING3 turnover and enhanced ING3-stimulatd apoptosis upon UV irradiation. Taken together, ING3 is deregulated in melanomas as a result of both nucleus-to-cytoplasm shift and rapid degradation. The level of ING3 in the nucleus may be an important marker for human melanoma progression and prognosis. Restoration of ING3 expression significantly sensitizes melanoma cells to UV radiation through the activation of Fas/caspase-8 pathway.

The type II tumour suppressor ING3 has been shown to modulate transcription, cell cycle control, and apoptosis. To investigate the putative role of ING3 in melanoma development, we examined the expression of ING3 in 58 dysplastic nevi, 114 primary melanomas, and 50 metastatic melanomas with tissue microarray and immunohistochemistry. Overall ING3 was reduced in metastatic melanomas compared with dyslastic nevi and primary melanomas. Reduced nuclear ING3 staining also correlated with melanoma progression, increased cytoplasmic ING3 level, tumour location at sun-exposed sites, and a poorer disease-specific 5-year survival of patients with primary melanoma. Multivariate analysis revealed that nuclear ING3 staining can independently predict patient outcome in primary melanomas. In melanoma cells, ING3 expression was rapidly induced by UV irradiation. Using stable clones of melanoma cells overexpressing ING3, we showed that ING3 significantly promoted UV-induced apoptosis. Unlike its homologues ING1b and ING2, ING3-enhanced apoptosis upon UV irradiation was independent of functional p53. Furthermore, ING3 did not affect the expression of mitochondrial proteins but increased the cleavage of Bid and caspases. Moreover, ING3 upregulated Fas expression and ING3-mediated apoptosis was blocked by inhibiting caspase-8 or Fas activation. Knockdown of ING3 expression decreased UV-induced apoptosis remarkably, suggesting that ING3 plays a crucial role in cellular response to UV radiation. To explore how ING3 is deregulated in advanced melanomas, we examined ING3 expression in metastatic melanoma cells and found that ING3 was downregulated due to a rapid protein turnover in these cells. Further studies demonstrated that ING3 undergoes degradation via the ubiquitin-proteasome pathway. We also demonstrate that ING3 interacts with the SCF (Skp1/Cul1/Roc1/Skp2) E3 ligase complex. Knockdown of Cul1 or Skp2 significantly stabilized ING3 in melanoma cells. In addition, lysine residue 96 is essential for ING3 ubiquitination as its mutation to arginine completely abrogated ING3 turnover and enhanced ING3-stimulatd apoptosis upon UV irradiation. Taken together, ING3 is deregulated in melanomas as a result of both nucleus-to-cytoplasm shift and rapid degradation. The level of ING3 in the nucleus may be an important marker for human melanoma progression and prognosis. Restoration of ING3 expression significantly sensitizes melanoma cells to UV radiation through the activation of Fas/caspase-8 pathway.

The type II tumour suppressor ING3 has been shown to modulate transcription, cell cycle control, and apoptosis. To investigate the putative role of ING3 in melanoma development, we examined the expression of ING3 in 58 dysplastic nevi, 114 primary melanomas, and 50 metastatic melanomas with tissue microarray and immunohistochemistry. Overall ING3 was reduced in metastatic melanomas compared with dyslastic nevi and primary melanomas. Reduced nuclear ING3 staining also correlated with melanoma progression, increased cytoplasmic ING3 level, tumour location at sun-exposed sites, and a poorer disease-specific 5-year survival of patients with primary melanoma. Multivariate analysis revealed that nuclear ING3 staining can independently predict patient outcome in primary melanomas. In melanoma cells, ING3 expression was rapidly induced by UV irradiation. Using stable clones of melanoma cells overexpressing ING3, we showed that ING3 significantly promoted UV-induced apoptosis. Unlike its homologues ING1b and ING2, ING3-enhanced apoptosis upon UV irradiation was independent of functional p53. Furthermore, ING3 did not affect the expression of mitochondrial proteins but increased the cleavage of Bid and caspases. Moreover, ING3 upregulated Fas expression and ING3-mediated apoptosis was blocked by inhibiting caspase-8 or Fas activation. Knockdown of ING3 expression decreased UV-induced apoptosis remarkably, suggesting that ING3 plays a crucial role in cellular response to UV radiation. To explore how ING3 is deregulated in advanced melanomas, we examined ING3 expression in metastatic melanoma cells and found that ING3 was downregulated due to a rapid protein turnover in these cells. Further studies demonstrated that ING3 undergoes degradation via the ubiquitin-proteasome pathway. We also demonstrate that ING3 interacts with the SCF (Skp1/Cul1/Roc1/Skp2) E3 ligase complex. Knockdown of Cul1 or Skp2 significantly stabilized ING3 in melanoma cells. In addition, lysine residue 96 is essential for ING3 ubiquitination as its mutation to arginine completely abrogated ING3 turnover and enhanced ING3-stimulatd apoptosis upon UV irradiation. Taken together, ING3 is deregulated in melanomas as a result of both nucleus-to-cytoplasm shift and rapid degradation. The level of ING3 in the nucleus may be an important marker for human melanoma progression and prognosis. Restoration of ING3 expression significantly sensitizes melanoma cells to UV radiation through the activation of Fas/caspase-8 pathway. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

ING2 et ING3 (Inhibitor of Growth 2 and 3) sont des protéines suppressives de tumeurs appartenant à une famille de 5 protéines ING1 à ING5. Le travail de thèse a consisté à étudier l’implication des protéines ING2 et ING3 dans la réponse aux dommages à l’ADN. Les fonctions d’ING3 en tant que gène suppresseur de tumeur sont peu connues. L’étude principale a été d’analyser l’impact de l’inhibition d’ING3 sur la réponse aux cassures double brins de l’ADN. De plus, une étude antérieure de l’équipe a observé que l’inhibition de la protéine ING2 était associée à l’accumulation de H2AX, un marqueur des cassures double brins de l’ADN. Ainsi nous avons également cherché à savoir si ING2 pouvait jouer un rôle dans la signalisation et la réparation des cassures double brins de l’ADN. Nous montrons pour la première fois un rôle d’ING3 dans la signalisation et la réparation des cassures double brins. En effet, ING3 joue un rôle crucial dans la signalisation des cassures permettant la phosphorylation et l’activation de la kinase ATM. En accord avec ces fonctions, ING3 est impliquée dans la réparation des cassures double brins par NHEJ et HR ainsi que dans la recombinaison de classe des immunoglobulines. Nous avons également montré l’implication d’ING2 dans la réponse aux cassures double brins de l’ADN. En effet, ING2 est nécessaire pour le recrutement de la protéine médiatrice de la réponse aux dommages 53BP1. Nos travaux montrent que ING2 est nécessaire pour la réparation par le mécanisme de la NHEJ. L’étude de son implication dans le mécanisme de recombinaison de classe des immunoglobulines montre qu’ING2 est un acteur essentiel de la voie classique de la NHEJ. Ces travaux identifient, pour la première fois, une fonction de type « caretaker » pour ING3 dans la réponse aux cassures doubles brins. Nous montrons une nouvelle fonction de type « caretaker » pour ING2 qui joue un rôle dans la stabilité du génome via son implication dans la réponse aux cassures double brins de l’ADN. / ING2 and ING3 (Inhibitor of Growth 2 and 3) are tumor suppressor proteins belonging to the ING family (ING1 to ING5). The aim of my research project was to analyze the involvement of ING2 and ING3 proteins in response to DNA damages. The functions of ING3 as a tumor suppressor gene are little known. In the present study, we have investigated the impact of ING3 inhibition in response to DNA double strand breaks. Previous study in the lab showed . In addition, a previous study in the lab found that inhibition of ING2 protein is associated with the accumulation of H2AX, a marker of DNA double-strand breaks. Thus, we also demonstrate that ING2 plays a role in the signaling and repair of DNA double-strand breaks. In the present study, we describe for the first time the involvement of ING3 in the signaling and repair of DNA double-strand breaks. ING3 allowed the phosphorylation and activation of the ATM kinase and the repair of double strand breaks by NHEJ and HR as well as in immunoglobulin class switch recombination. We also show the involvement of ING2 in this process. Indeed, ING2 is necessary for 53BP1 recruitment in response to DNA damages and repair by the mechanism of NHEJ. ING2 was also an essential actor for the class switch recombination demonstrated that ING2 is an essential actor of the classical NHEJ pathway. This work identifies, for the first time, a "caretaker" function for ING3 in the response to DNA double strand breaks; and . We show a new caretaker function for ING2 that plays a role in the stability of the genome through its involvement in DNA damage response.

Les gènes ING (Inhibitor of Growth) sont des gènes candidats suppresseurs de tumeurs conservés de la Levure à l'Homme. Les protéines ING ont des fonctions suppressives de tumeurs de type I ou « caretaker » car elles participent aux processus de maintien de la stabilité du génome en régulant la réplication et la réparation de l'ADN. Elles ont aussi des fonctions suppressives de tumeurs de type II ou « gatekeeper » puisqu'elles sont impliquées dans la régulation de la prolifération cellulaire de façon dépendante et indépendante de p53 et car elles contrôlent la transcription génique en participant au remodelage de la chromatine. L'objectif de ma thèse est de mieux comprendre l'implication de ING1, ING2 et ING3 dans les voies de suppression des tumeurs. Nos travaux montrent que ING1 est sumoylée sur la lysine 193 principalement par l'E3 SUMO ligase PIAS4, afin de réguler l'ancrage de ING1 sur le promoteur de gènes cibles pour réguler leur transcription. Nous avons aussi décrit pour la première fois l'implication de ING2 et de ING3 dans la réponse aux cassures double brin de l'ADN. Nous montrons que cette fonction est conservée entre ING2, ING3 et leur orthologues, respectivement, Pho23 et Yng2 chez la Levure Saccharomyces cerevisiae. ING2 contrôle l'accumulation de PIAS4 au niveau des sites de dommages et régule la sumoylation de l'E3 ubquitine ligase RNF168, afin de permettre la signalisation et la réparation des cassures double brin de l'ADN. ING3 est nécessaire à l'accumulation de 53BP1 et contrôle la réparation de ces dommages. Ces travaux contribuent donc à une meilleure connaissance du rôle des ING dans les voies de suppression des tumeurs. Ils permettent de mieux comprendre comment ING1 régule la transcription génique et décrivent une nouvelle fonction suppressive de tumeur de type I ou « caretaker » pour ING2 et ING3 dans le maintien de la stabilité du génome. / ING (Inhibitor of Growth) genes are tumor suppressor gene candidates conserved from Yeast to Humans. ING proteins have type I tumor suppressive functions or "caretaker" because they participate in the maintenance of genome stability by regulating DNA replication and repair processes. They have also tumor suppressive functions of type II or "gatekeeper" because they are involved in the regulation of cell proliferation in p53 dependent and independent manners. They also participate in the regulation of gene transcription by regulating chromatin remodeling. The aim of my thesis was to better understand how ING1, ING2 and ING3 are involved in tumor suppressive pathways. Our work shows that ING1 is sumoylated on lysine 193 mainly by the SUMO E3 ligase PIAS4 to regulate ING1 anchoring on target gene promoters to control gene transcription. We have also described the involvement of ING2 and ING3 in the DNA double strand breaks response. We show the conservation of this function between ING2, ING3 and their orthologs, respectively, Pho23 and Yng2 in Yeast Saccharomyces cerevisiae. ING2 controls the accumulation of PIAS4 at DNA damage sites and regulates the sumoylation of the E3 ubiquitin ligase RNF168, to regulate DNA double strand break signaling and repair. ING3 is necessary for the accumulation of 53BP1 and promotes DNA damage repair. This work contributes to a better understanding of the role of ING proteins in tumor suppression. It thus provides new insights of how ING1 regulates gene transcription and emphasizes a new tumor suppressive function of type I or "caretaker" for ING2 and ING3 in the genome stability maintenance.