Le cancer gastrique (CG) est traité par résection chirurgicale combinée à une chimiothérapie à base de composés de platine. La résistance croissante aux chimiothérapies renforce la nécessité d’identifier des marqueurs moléculaires robustes pour adapter le traitement et développer des thérapies ciblées. Durant ma thèse, j’ai montré l’importance des voies de l’épigénétique dans le mode d’action de drogues anti-cancéreuses dans le CG. Notamment, j’ai identifié le rôle d’une histone déacétylase, HDAC4, et de plusieurs miRNAs, dont miR-140, dans la réponse au cisplatine. De plus, j’ai démontré que des composés à base de ruthénium ayant des propriétés redox agissent indépendant de l’ADN et de p53 mais affectent certaines régulations épigénétiques. Ceci m’a donc conduit à étudier l’intérêt thérapeutique et les mécanismes sous-jacents d’un traitement combiné associant le cisplatine et des inhibiteurs de HDAC. L’ensemble de ces résultats permet d’ouvrir de nouvelles perspectives dans la compréhension des mécanismes d’action des drogues anticancéreuses dans le CG et dans l’identification de marqueurs pronostiques ou de thérapie innovante plus adaptée. / Gastric cancer (GC) is treated by surgical resection combined with chemotherapy based on platinum compounds. The increase in chemotherapy resistance reinforces the need to identify robust molecular markers to tailor treatment and develop targeted therapies. During my PhD, I examined the importance of the epigenetic pathways in the mode of action of anticancer drugs in gastric cancer. In particular, I have identified the role of one histone deacetylase, HDAC4, and several miRNAs, including miR-140, in response to cisplatin. Moreover, I have shown that ruthenium compounds having redox properties act independently of DNA and p53 but affect some epigenetic regulations. This then led me to investigate the therapeutic value and the underlying mechanisms of a combined therapy associating cisplatin and HDAC inhibitors. All these results will open new perspectives in the understanding of the mechanisms of action of anticancer drugs in gastric cancer and in the identification of prognostic markers or more appropriate advanced therapy.

Molecular defects in the p63 gene give rise to severe physiological abnormalities in patients with ectodermal dysplasia, however the mechanisms by which p63 mutations disrupt p63 function are unknown. In this study we examined four ΔNp63α mutants; Ectrodactyly-Ectodermal Dysplasia with Clefting (EEC) R204W, R304W and Ankyloblepharon-Ectodermal Dysplasia with Clefting (AEC) mutants, L514F and G530V, and characterized DNA binding, transcription factor activity, oligomerization with wild-type p63 and changes in protein stability/nuclear localization. We also investigated the putative OD-SAM interaction in p63 and p73. We demonstrated that both the EEC and AEC mutants cannot transcriptionally activate the PERP promoter and can hetero-oligomerize forming dominant negative complexes with wild-type p63. We show that both EEC mutants and AEC L514F mutants are more stable which is not due to aberrant degradation by the E3 ligase Itch. Finally, we discovered that a novel interaction between the p73 OD and SAM domain is absent in p63.

Molecular defects in the p63 gene give rise to severe physiological abnormalities in patients with ectodermal dysplasia, however the mechanisms by which p63 mutations disrupt p63 function are unknown. In this study we examined four ΔNp63α mutants; Ectrodactyly-Ectodermal Dysplasia with Clefting (EEC) R204W, R304W and Ankyloblepharon-Ectodermal Dysplasia with Clefting (AEC) mutants, L514F and G530V, and characterized DNA binding, transcription factor activity, oligomerization with wild-type p63 and changes in protein stability/nuclear localization. We also investigated the putative OD-SAM interaction in p63 and p73. We demonstrated that both the EEC and AEC mutants cannot transcriptionally activate the PERP promoter and can hetero-oligomerize forming dominant negative complexes with wild-type p63. We show that both EEC mutants and AEC L514F mutants are more stable which is not due to aberrant degradation by the E3 ligase Itch. Finally, we discovered that a novel interaction between the p73 OD and SAM domain is absent in p63.

Introdução: O gene p63 é o mais ancentral membro dos integrantes da família p53 e é descrito como o responsável pela manutenção da integridade gênica e regulação do ciclo celular em células germinativas imaturas. Porém, ainda não há relatos na literatura que avaliem sua expressão em células da granulosa luteinizadas. A endometriose é uma doença crônica que está associada à infertilidade. Vários mecanismos foram propostos para explicar esta associação, mas, até o momento, nenhum deles é considerado definitivo. Objetivos: O objetivo desse estudo é avaliar a expressão do gene e da proteína p63 em células da granulosa luteinizadas de pacientes inférteis submetidas à fertilização in vitro (IVF). Além disso, verificar se pacientes com endometriose apresentam função anormal da p63. Métodos: Realizamos um estudo transversal e prospectivo. Nós coletamos células da granulosa de 28 pacientes submetidas a IVF para avaliar a expressão de p63. Depois, para estudar o efeito na endometriose dividimos em dois grupos: (1) grupo estudo (n=9): pacientes com diagnóstico videolaparoscópico de endometriose e (2) grupo controle (n=19): pacientes inférteis por outros motivos, sem endometriose. As células coletadas foram preparadas e analisadas para expressão gênica (PCR em tempo real) e expressão proteica (imunofluorescência). Os resultados foram comparados entre os grupos. Resultados: Não observamos diferença significativa de expressão do gene p63 entre os grupos estudados. A mediana do 2-ΔΔCT no grupo controle foi 0,93 (IC 95%, 0,55- 2,83) e 0,88 no grupo em estudo (IC 95%, 0,24-2,84). O resultado também mostrou que o gene p63 não é expresso nos dois grupos. Quanto à expressão da proteína p63, a imunofluorescência não demonstrou expressão em nenhum dos dois grupos. Conclusão: O gene e a proteína p63 podem ser muito importantes na manutenção da integridade gênica de folículos imaturos de reserva, não dependentes de FSH. Porém, após o recrutamento e crescimento folicular, não se observa mais sua expressão, ou seja, ele não faz parte da maturação e desenvolvimento oocitário. / Introduction: The p63 gene is the most ancient member of the components of p53 family and is described as the responsible for maintaining of genic integrity and cell cycle regulation in immature germ cells. However, there are no reports in the literature evaluating its expression in granulosa cells luteinized. Endometriosis is a chronic disease that is associated with infertility. Several mechanisms have been proposed to explain this association, but so far, none of them is considered definitive. Objectives: The purpose of this study is to evaluate the expression of the p63 gene and protein in granulosa luteinized cells of infertile patients undergoing in vitro fertilization (IVF). Also, we checked whether patients with endometriosis have abnormal function of p63. Methods: We performed a prospective cross-sectional study. We collected granulosa cells of 28 patients undergoing IVF to evaluate p63 expression. Then, to study the effect on endometriosis, they were divided into two groups: (1) study group (n = 9): patients with laparoscopic diagnosis of endometriosis and (2) control group (n = 19): infertile patients for other reasons, without endometriosis . The collected cells were prepared and analyzed for gene expression (real time PCR) and protein expression (immunofluorescence). The results were compared between the groups. Results: There was no significant difference in expression of the p63 gene between the groups. The median of 2-ΔΔCT in the the control group was 0.93 (95% CI, 0.55 to 2.83) and 0.88 in the study group (95% CI, 0.24 to 2.84). The results also showed that the p63 gene is not expressed in granulosa cells in both groups. About to the p63 protein expression, immunofluorescence showed no expression in either group. Conclusion: The p63 gene and protein can be very important in maintaining the genic integrity of immature reserve follicles, not dependent of FSH. However, after the recruitment and follicular growth is not more observed its expression, suggesting that p63 is not part of control of oocyte maturation and development.

L'atrophie musculaire de la cachexie provient du déséquilibre entre la synthèse et la dégradation des protéines. La littérature suggère que les membres de la famille p53 (p53, p63, p73) jouent un rôle dans le contrôle des processus de prolifération, différenciation et mort des précurseurs et des fibres musculaires. Ici nous avons caractérisé le profil d'expression de ces membres dans l'atrophie musculaire de la SLA (Sclérose Latérale Amyotrophique) et dans un modèle de cachexie induite par la doxorubicine. Nous avons montré une augmentation de l'expression des membres de la famille p53 et des atrogènes de manière corrélée sur ces deux modèles ainsi qu’une activation transcriptionnelle de Trim63 par p53, p63 et p73. Aussi, nous avons voulu savoir si les composés de tocophérol possédant une activité antioxydante pouvait réduire l'atrophie musculaire et avons montré que ce composé neutralise l'induction de la voie Notch, importante pour le développement musculaire et la régénération. / Muscle atrophy in cachexia results from the imbalance between protein synthesis and degradation due to activation of the ubiquitin-proteasome pathway. Literature suggests that p53 family members play a role in controlling proliferation, differentiation and death of precursors and muscle fibers. Here we characterize the expression profile of the p53 family members in muscle atrophy in ALS (Amyotrophic Lateral Sclerosis) and in doxorubicin induced cachexia model. We revealed an increased expression of the p53 family members and atrogenes in a correlated manner on both models and a transcriptional activation of Trim63 by p53, p63 and p73. Importantly, we also show that ROS and ceramide accumulation are important for Trim63 induction by doxorubicin. In addition, we tested whether compounds of tocopherol harboring antioxidant activity might reduce muscle atrophy. We showed that this compound counteracts the induction of the Notch pathway, important to muscle development and regeneration.

Introdução: O gene p63 é o mais ancentral membro dos integrantes da família p53 e é descrito como o responsável pela manutenção da integridade gênica e regulação do ciclo celular em células germinativas imaturas. Porém, ainda não há relatos na literatura que avaliem sua expressão em células da granulosa luteinizadas. A endometriose é uma doença crônica que está associada à infertilidade. Vários mecanismos foram propostos para explicar esta associação, mas, até o momento, nenhum deles é considerado definitivo. Objetivos: O objetivo desse estudo é avaliar a expressão do gene e da proteína p63 em células da granulosa luteinizadas de pacientes inférteis submetidas à fertilização in vitro (IVF). Além disso, verificar se pacientes com endometriose apresentam função anormal da p63. Métodos: Realizamos um estudo transversal e prospectivo. Nós coletamos células da granulosa de 28 pacientes submetidas a IVF para avaliar a expressão de p63. Depois, para estudar o efeito na endometriose dividimos em dois grupos: (1) grupo estudo (n=9): pacientes com diagnóstico videolaparoscópico de endometriose e (2) grupo controle (n=19): pacientes inférteis por outros motivos, sem endometriose. As células coletadas foram preparadas e analisadas para expressão gênica (PCR em tempo real) e expressão proteica (imunofluorescência). Os resultados foram comparados entre os grupos. Resultados: Não observamos diferença significativa de expressão do gene p63 entre os grupos estudados. A mediana do 2-ΔΔCT no grupo controle foi 0,93 (IC 95%, 0,55- 2,83) e 0,88 no grupo em estudo (IC 95%, 0,24-2,84). O resultado também mostrou que o gene p63 não é expresso nos dois grupos. Quanto à expressão da proteína p63, a imunofluorescência não demonstrou expressão em nenhum dos dois grupos. Conclusão: O gene e a proteína p63 podem ser muito importantes na manutenção da integridade gênica de folículos imaturos de reserva, não dependentes de FSH. Porém, após o recrutamento e crescimento folicular, não se observa mais sua expressão, ou seja, ele não faz parte da maturação e desenvolvimento oocitário. / Introduction: The p63 gene is the most ancient member of the components of p53 family and is described as the responsible for maintaining of genic integrity and cell cycle regulation in immature germ cells. However, there are no reports in the literature evaluating its expression in granulosa cells luteinized. Endometriosis is a chronic disease that is associated with infertility. Several mechanisms have been proposed to explain this association, but so far, none of them is considered definitive. Objectives: The purpose of this study is to evaluate the expression of the p63 gene and protein in granulosa luteinized cells of infertile patients undergoing in vitro fertilization (IVF). Also, we checked whether patients with endometriosis have abnormal function of p63. Methods: We performed a prospective cross-sectional study. We collected granulosa cells of 28 patients undergoing IVF to evaluate p63 expression. Then, to study the effect on endometriosis, they were divided into two groups: (1) study group (n = 9): patients with laparoscopic diagnosis of endometriosis and (2) control group (n = 19): infertile patients for other reasons, without endometriosis . The collected cells were prepared and analyzed for gene expression (real time PCR) and protein expression (immunofluorescence). The results were compared between the groups. Results: There was no significant difference in expression of the p63 gene between the groups. The median of 2-ΔΔCT in the the control group was 0.93 (95% CI, 0.55 to 2.83) and 0.88 in the study group (95% CI, 0.24 to 2.84). The results also showed that the p63 gene is not expressed in granulosa cells in both groups. About to the p63 protein expression, immunofluorescence showed no expression in either group. Conclusion: The p63 gene and protein can be very important in maintaining the genic integrity of immature reserve follicles, not dependent of FSH. However, after the recruitment and follicular growth is not more observed its expression, suggesting that p63 is not part of control of oocyte maturation and development.

Introdução: O gene p63 é o mais ancentral membro dos integrantes da família p53 e é descrito como o responsável pela manutenção da integridade gênica e regulação do ciclo celular em células germinativas imaturas. Porém, ainda não há relatos na literatura que avaliem sua expressão em células da granulosa luteinizadas. A endometriose é uma doença crônica que está associada à infertilidade. Vários mecanismos foram propostos para explicar esta associação, mas, até o momento, nenhum deles é considerado definitivo. Objetivos: O objetivo desse estudo é avaliar a expressão do gene e da proteína p63 em células da granulosa luteinizadas de pacientes inférteis submetidas à fertilização in vitro (IVF). Além disso, verificar se pacientes com endometriose apresentam função anormal da p63. Métodos: Realizamos um estudo transversal e prospectivo. Nós coletamos células da granulosa de 28 pacientes submetidas a IVF para avaliar a expressão de p63. Depois, para estudar o efeito na endometriose dividimos em dois grupos: (1) grupo estudo (n=9): pacientes com diagnóstico videolaparoscópico de endometriose e (2) grupo controle (n=19): pacientes inférteis por outros motivos, sem endometriose. As células coletadas foram preparadas e analisadas para expressão gênica (PCR em tempo real) e expressão proteica (imunofluorescência). Os resultados foram comparados entre os grupos. Resultados: Não observamos diferença significativa de expressão do gene p63 entre os grupos estudados. A mediana do 2-ΔΔCT no grupo controle foi 0,93 (IC 95%, 0,55- 2,83) e 0,88 no grupo em estudo (IC 95%, 0,24-2,84). O resultado também mostrou que o gene p63 não é expresso nos dois grupos. Quanto à expressão da proteína p63, a imunofluorescência não demonstrou expressão em nenhum dos dois grupos. Conclusão: O gene e a proteína p63 podem ser muito importantes na manutenção da integridade gênica de folículos imaturos de reserva, não dependentes de FSH. Porém, após o recrutamento e crescimento folicular, não se observa mais sua expressão, ou seja, ele não faz parte da maturação e desenvolvimento oocitário. / Introduction: The p63 gene is the most ancient member of the components of p53 family and is described as the responsible for maintaining of genic integrity and cell cycle regulation in immature germ cells. However, there are no reports in the literature evaluating its expression in granulosa cells luteinized. Endometriosis is a chronic disease that is associated with infertility. Several mechanisms have been proposed to explain this association, but so far, none of them is considered definitive. Objectives: The purpose of this study is to evaluate the expression of the p63 gene and protein in granulosa luteinized cells of infertile patients undergoing in vitro fertilization (IVF). Also, we checked whether patients with endometriosis have abnormal function of p63. Methods: We performed a prospective cross-sectional study. We collected granulosa cells of 28 patients undergoing IVF to evaluate p63 expression. Then, to study the effect on endometriosis, they were divided into two groups: (1) study group (n = 9): patients with laparoscopic diagnosis of endometriosis and (2) control group (n = 19): infertile patients for other reasons, without endometriosis . The collected cells were prepared and analyzed for gene expression (real time PCR) and protein expression (immunofluorescence). The results were compared between the groups. Results: There was no significant difference in expression of the p63 gene between the groups. The median of 2-ΔΔCT in the the control group was 0.93 (95% CI, 0.55 to 2.83) and 0.88 in the study group (95% CI, 0.24 to 2.84). The results also showed that the p63 gene is not expressed in granulosa cells in both groups. About to the p63 protein expression, immunofluorescence showed no expression in either group. Conclusion: The p63 gene and protein can be very important in maintaining the genic integrity of immature reserve follicles, not dependent of FSH. However, after the recruitment and follicular growth is not more observed its expression, suggesting that p63 is not part of control of oocyte maturation and development.

No description available.

Le gène TP63 fait partie de la famille du gène suppresseur de tumeur TP53. Il code plusieurs isoformes. L’une d’entre elles, tronquée dans la région amino-terminale et appelée ΔNp63, présente des propriétés oncogéniques. Elle est impliquée dans la progression tumorale et la chimiorésistance. Sa surexpression est fréquente dans certains types de cancers. La première partie de mes travaux a consisté à identifier les facteurs de transcription impliqués dans la régulation du promoteur de ΔNp63. J’ai montré que l’expression de cette isoforme est inhibée par p53 et activée par ΔNp63 elle-même et par la β-caténine, dans des lignées de carcinome hépatocellulaire et de carcinome épidermoïde de l’œsophage. Dans des conditions physiologiques, un des types cellulaires dans lequel ΔNp63 est exprimée est la cellule basale mammaire. Il est admis que les tumeurs mammaires dites basal-like sont issues des cellules basales. Certaines de ces tumeurs présentant une surexpression de ΔNp63, nous avons émis l’hypothèse que ΔNp63 serait impliquée dans la tumorigenèse des cellules basales. Dans la deuxième partie, j’ai montré que l’expression de ΔNp63 peut être augmentée en cultivant les cellules mammaires en présence de surnageant de fibroblastes embryonnaires humains. L’identification de facteurs solubles responsables est en cours. D’autre part, j’ai caractérisé les conséquences biologiques de cette augmentation en termes de prolifération, de motilité et de survie des cellules immatures mammaires normales et tumorales. Les plus grandes modifications observées concernent (i) l’état de différenciation, les cellules surexprimant ΔNp63 présentant un phénotype plus immature ; (ii) la balance entre migration et adhésion qui penche en faveur de cette dernière. ΔNp63 semble donc être au carrefour des mécanismes de prolifération, d’adhésion, de différenciation et de motilité, processus impliqués dans la formation et l’homéostasie des tissus, mais dont l’altération peut conduire à l’initiation et à la progression tumorale ainsi qu’à la dissémination métastatique. Mes travaux apportent des informations sur le rôle de cette protéine dans ces processus et devraient, à terme, permettre de mieux comprendre la genèse de certains cancers, en particulier les carcinomes basal-like / TP63 gene belongs to the TP53 tumor suppressor gene family. It encodes several isoforms. One of these, truncated in its amino-terminal end and called ΔNp63, displays oncogenic properties. It is involved in tumor progression and chemoresistance and is overexpressed in some tumor types. The first part of my work consisted of identifying the transcription factors involved in the regulation of the ΔNp63 promoter. I have shown that ΔNp63 expression is inhibited by p53 and activated by ΔNp63 itself and by β-catenin in hepatocellular carcinoma and esophageal squamous cell carcinoma cell lines. Under physiological conditions, one of the cell types in which ΔNp63 is expressed is the mammary basal cell. The “basal-like” mammary tumor sub-type seems to stem from basal cells. As some of these tumors exhibit overexpression of ΔNp63, we hypothesized that this isoform could be involved in the genesis of basal-like tumors. In the second part, I have shown that ΔNp63 expression can be increased in mammary cells cultivated in the presence of human embryonic fibroblast supernatant. Identifying the soluble factors responsible for this increase is in progress. In parallel, I have evaluated the biological consequences of ΔNp63 overexpression in terms of proliferation, cell motility and survival of normal and malignant immature mammary cells. The main modifications relate to (i) the differentiation status, ΔNp63-overexpressing cells exhibiting a more immature phenotype; (ii) the balance between migration and adhesion that is in favor of this latter. ΔNp63 seems to be at the crossroads of proliferation, adhesion, differentiation and motility, processes implicated in tissue formation and homeostasis, but whose alteration may lead to tumor initiation and progression and to metastatic dissemination. My work provides information on the role of this isoform in these processes and should allow better understanding of the genesis of some tumor types, in particular basal-like breast carcinomas

Ph.D. / Molecular and Medical Genetics / The objective of this study is to examine, in light of the expression of multiple p53 family member isoforms, the specific role of p73 in malignant conversion, cellular response to DNA damage, and direct or indirect cooperation with other p53 family members in a clonal model of epidermal carcinogenesis. We first focused on the role of p73 in malignant conversion. Whether sporadic or siRNA induced, loss of p73 in initiated p53+/+ keratinocytes lead to conversion to squamous cell carcinoma (SCC) in vivo which was reversible upon reconstitution of TAp73α but not ΔNp73α. Second, we investigated the cellular response to ionizing radiation (IR) in the presence and absence of p73, showing that loss of p73 at malignant conversion was associated with resistance to IR in vitro. The loss of radiation sensitivity and malignant conversion was characterized by reduced steady state DNA binding levels of transcriptionally active p63 isoforms to the p21 promoter, failure to induce specific p53 family transcriptional targets, and failure to arrest in G1. Reconstitution of TAp73α, but not ΔNp73α, increased steady state DNA binding capabilities of TAp63β, TAp63γ, and ΔNp63γ, and steady state levels of p53 family target mRNA, but did not restore cellular sensitivity to IR. We thus uncovered a functional cooperation between TA isoforms of p73 and p63 and showed that p73-mediated DNA damage response was uncoupled from its tumor suppressive role. We observed preferential DNA binding of the inhibitory ΔNp63α isoform both in vitro and invivo in SCC suggesting that in the absence of TAp73α a balance is tipped toward DNA binding of the inhibitory isoforms. Third, we studied the role of the p53 family inkeratinocyte response to UVB. Tumorigenic cells lacking p73 that were resistant to IR remained sensitive to UVB, accompanied by DNA binding of the TAp63γ isoform, suggesting that keratinocyte response to UVB is not dependent upon p73 and suggesting a hierarchy of p53 family member responses to DNA damage. Finally, we examined TAp73α interaction with the p53 family inhibitor Mdm2. Mdm2 was in complex with DNA-bound p53 family members in malignant cells, but reconstitution of cells withTAp73α correlated with removal of Mdm2 from the complex, making them more like primary keratinocytes or initiated cells. Like the initiated cells, cells expressing TAp73α were refractory to treatment with the Mdm2-p53 inhibitor Nutlin-3 while cells lacking p73 expression or expressing ΔNp73α were sensitive. Thus, we suggest that p73 may be acting as a molecular shield to keep p53 family member inhibitors, such as ΔNp63α andMdm2, at bay. Further understanding of p53 family interplay in tumor development and DNA damage response could lead to new therapies or optimization of current therapeutic strategies in solid tumors of epithelium, particularly where deregulation or loss of p63 and p73 expression is associated with increased tumor invasiveness, treatment resistance, and poor patient prognosis.