Bromodomain protein 4 (Brd4) plays critical roles in development, cancer progression and virus-host pathogenesis. Papillomaviruses (PV) E2 protein associates with Brd4 and this interaction is important for transcriptional regulation of the viral oncogenes by E2 as well as viral genome maintenance in host cells for some of the PV. Brd4 is causally linked to a rare, aggressive cancer, NUT Midline Carcinoma (NMC), which is typically defined by chromosomal translocation fusing the NUT gene to the Brd4 gene. The molecular mechanism behind Brd4-NUT oncogenesis remains largely unknown. To gain mechanistic insight into the biological functions of Brd4, we performed a proteomic analysis to identify and characterize Brd4 associated cellular proteins. We discovered binding partners of the Brd4 ET domain and show that interaction of these proteins with Brd4 is conserved across the human BET proteins. The Brd4 ET interactors, NSD3, JMJD6 and GLTSCR1, were found to be important for Brd4 transcriptional activation function and are recruited to the promoters they regulate in a Brd4 dependent manner. Moreover, depletion of Brd4 or NSD3 reduced H3K36 methylation demonstrating that the Brd4/NSD3 complex regulates the chromatin microenvironment. We thus identified the ET domain as an important transcription regulatory domain for Brd4. Since the ET domain is preserved in the Brd-NUT proteins, we also investigated its contribution to Brd-NUT pathogenesis. Expression of the ET domain, which competes off the ET domain interactors from Brd4-NUT, induced squamous differentiation. More specifically, depletion of the ET domain interactor, NSD3 induced squamous differentiation by Brd4-NUT while loss of JMJD6 markedly reduced proliferation of the NMC cells. Lastly, we investigated the effect of the recently developed small molecule inhibitors of BET bromodomains on PV E2 functions and papilloma virus mediated pathogenesis. BET inhibitors blocked association of Brd4 and E2 with mitotic chromosomes without affecting Brd4 dependent E2 transcription regulation of viral promoters. This finding suggests that Brd4 affects viral genome maintenance and viral transcription regulation via different mechanisms. Overall, these studies have shed new insight into the molecular mechanism of Brd4 functions and their role in human diseases.

Les mastocytes humains (MC) sont des cellules tissulaires d’origine hématopoïétique impliquées dans une série de processus physiologiques et pathologiques. Les recherches sur les MC ont été entravées pendant longtemps en raison de l'accès limité à des populations pures de ces cellules. Nous avons établi une nouvelle lignée humaine de MC, ROSAKIT WT, dont les propriétés sont similaires à celles des MC primaires, constituant un nouvel outil pour la recherche sur les fonctions des MC humains, et permettant le criblage à haut débit de thérapies anti-allergiques. Les MC sont impliqués dans les mastocytoses, où ils s’accumulent pathologiquement dans divers tissus. Bien que la plupart des cas de mastocytoses systémiques (SM) sont chroniques et indolents, les patients atteints de SM avancée (SM agressive; ASM, et leucémie à mastocytes; MCL) ont un mauvais pronostic, car la plupart des thérapies disponibles ne sont pas curatives. Afin de mieux comprendre la physiopathologie des formes avancées de SM et pour trouver de nouvelles approches pour le traitement, nous avons profité de la disponibilité des cellules ROSAKIT WT pour établir un nouveau sous-clone, la lignée cellulaire ROSAKIT D816V, représentant un équivalent des cellules néoplasiques s’accumulant dans les SM. L'utilisation de cette lignée et de cellules des patients nous a permis d’identifier BRD4 comme une nouvelle cible thérapeutique dans les ASM et les MCL. Nous avons démontré que les MC néoplasiques de patients avec ASM expriment des quantités substantielles de BRD4. Fait intéressant, nous avons aussi démontré que les lignées cellulaires HMC-1 et ROSAKIT D816V expriment aussi BRD4, et que leur prolifération est inhibée par un shRNA BRD4-spécifique. En outre, nous avons montré que le médicament JQ1, inhibiteur de BRD4, induit une inhibition de la croissance et une apoptose dose-dépendante dans les mêmes cellules. De plus, nous avons démontré que JQ1 supprime également la prolifération des MC néoplasiques primaires de patients atteints d’ASM ou de MCL à de faibles concentrations. Enfin, nous avons observé que la midostaurine (PKC412) et l’acide rétinoïque tout-trans (ATRA) coopèrent avec JQ1 pour induire des effets inhibiteurs synergiques sur l’inhibition de la survie des mêmes cellules. En conclusion, nos résultats représentent une avancée sur ce qui était précédemment connu sur l’implication de BRD4 dans les mastocytoses et nous ont permis d'identifier cette protéine comme cible thérapeutique prometteuse dans le traitement des formes avancées de SM. / Human mast cells (MCs) are hematopoietic stem cell (HSC)-derived, tissue-resident, multifaceted cells involved in a myriad of physiological and pathological processes. Researches on MCs have been hampered for a long time, due to limited access to pure populations of these cells. We have established a new human MC line, ROSAKIT WT, whose properties are similar to those of primary HSC-derived MCs, providing a novel tool for research on human MC functions, and enabling the high-throughput screening of anti-allergic therapies. Among others, MCs are involved in a group of diseases termed mastocytosis, where they accumulate pathologically in various tissues. Although most cases of systemic mastocytosis (SM) are chronic with an indolent course, patients with advanced SM (aggressive SM; ASM, and mast cell leukemia; MCL) have a reduced life expectancy and a poor prognosis, since most of the therapies already available are not curative. In order to better understand the pathophysiology of advanced SM and to. find new approaches for treatment, we took advantage of the availability of the ROSAKIT WT cells to establish a new subclone, the ROSAKIT D816V cell line, representing a paradigm of the neoplastic cells accumulating in SMUsing these malignant cell line and patients’ cells, we identified the epigenetic reader bromodomain-containing protein-4 (BRD4) as a novel drug target in ASM and MCL. Indeed, we demonstrated that neoplastic MCs from ASM patients expressed substantial amounts of BRD4. Interestingly, we then demonstrated that HMC-1 and ROSAKIT D816V cell lines express BRD4, and that their proliferation is inhibited by a BRD4-specific shRNA. Moreover, we showed that the BRD4-targeting drug JQ1 induced a dose-dependent growth inhibition and apoptosis in the same cells. In addition, we demonstrated that JQ1 suppressed also the proliferation of primary neoplastic MCs of patients with ASM or MCL at low concentrations. Finally, we reported that midostaurin (PKC412) and all-trans retinoic acid (ATRA) cooperated with JQ1 in producing synergistic inhibitory effects on the survival of HMC-1 and ROSA cells. Together, our data represent a significant advance over what was previously known on the involvement of BRD4 in mastocytosis and identify this epigenetic reader bromodomain-containing protein as a promising drug target in advanced SM

No description available.

Since the discovery of DNA packaging into chromatin, and McClintock's (1951) work on position-effect variegation providing evidence of non-mendelian inheritance, the principal of a genome maintaining 'on' and 'off' states has been widely adopted. However, the underlying mechanisms that regulate these dynamic chromatin states and their effect on disease are still poorly understood. DNA methylation and histone trimethylation at H3K9 and H4K20 are the core hallmarks of the heterochromatic constitutively 'off' state. Constitutive heterochromatin is predominantly comprised of repetitive satellite containing pericentromeric regions and telomeres and in mouse heterochromatin clusters into large chromocenters. These regions are cytologically more compact and generally transcriptionally silent across embryonic and differentiated mouse cell types. However, in addition to increased genomic instability, mouse tumour cells sustain increased satellite expression suggesting constitutive heterochromatin is disrupted. Therefore how constitutive heterochromatin is maintained has important implications for genome regulation and disease, and remains poorly understood. While satellite DNA sequences are not evolutionarily conserved, pericentromeric and telomeric heterochromatin occurs across species. Heterochromatin formation is therefore independent of the underlying DNA sequence, supporting the hypothesis that epigenetic components can regulate chromatin structure. DNA methylation is generally thought to be associated with transcriptional silencing and chromatin compaction. However, Gilbert et al (2007) showed that the complete loss of DNA methylation did not affect the compaction at heterochromatin or global genome compaction. The role of H3K9me3 in regulating heterochromatin has also been an area of keen interest. H3K9me3 patterns are established by suppressor of variegation 3-9 homologues and provide the binding site for heterochromatic protein 1 [HP1] which can in turn recruit Suv39h1. This Suv3-9h-HP1-H3K9 axis enables its propagation throughout heterochromatin. Peters et al (2001) demonstrated that in mice loss of suv39 homologues 1 and 2 caused a loss of H3K9me3 at constitutive heterochromatic domains. These Suv39h null mice demonstrated decreased genome stability, and an increased prevalence of oncogenesis. However cytological chromocenters are still present in the absence of H3K9me3. Therefore the function of H3K9me3 as a causative agent in heterochromatin formation is still debated. Broadly the aim was to investigate the phenotypic role of heterochromatic epigenetic components in cancer progression, and address whether H3K9me3 effects large scale chromatin structure. To identify heterochromatic gene silencing components, an inhibitor screen was performed in an artificial silenced reporter system. The reporter fluorophore was silenced by the presence of centromeric arrays from yeast/bacterial artificial chromosomes and human alpha satellite repeats enriched for H3K9me3. To address the function of the de-silencing components identified in cancer, the fitness of colon cancer cells [HCT116] was investigated before and after the development of resistance to the MEK inhibitor trametinib. The most intriguing result was that BET protein inhibition resulted in derepression of the reporter construct and trametinib resistant HCT116 cells were more sensitive to BET inhibitors, while subsequent investigation showed HP1 protein levels were altered. Analysis of publically available datasets of tumour drug resistance, showed elevated BET protein binding at HP1 promoters in resistant cell lines suggesting an indirect role in gene silencing. To investigate the consequence of H3K9me3 loss on chromatin structure, mouse embryonic stem cells that lacked both Suv39 homologues were used. Microccocal nuclease digestion and sucrose sedimentation demonstrated a global decompaction of large-scale chromatin fibres whilst re-expression of suv39h1 rescued H3K9me3 at chromocenters and global chromatin decompaction. Loss of Suv39h also increased chromatin associated RNA levels that were also rescued by Suv39h1 re-expression. This suggests that H3K9me3 has a role chromatin fibre compaction globally as well as at constitutive heterochromatin, potentially mediated by chromatin associated RNA. To conclude, multiple components were identified that are involved in transcriptional silencing. Evaluating their function in tumour progression demonstrated a possible role of BET proteins in the development of MEKi resistance that may be mediated through HP1 proteins. H3K9me3 and its binding partner HP1 affect global chromatin compaction. The global decompaction after Suv39h loss correlates with an increase in chromatin associated RNA, suggesting a possible mechanism for changes in chromatin compaction beyond H3K9me3.

Il apparait de nos jours évident que les cancers ne peuvent se réduire uniquement à des aberrations génétiques, et qu'un nouveau paramètre est à prendre en considération, l'épigénétique. Au cours de ma thèse je me suis efforcé de caractériser la protéine fusion BRD4-NUT. Cette protéine résulte d'une translocation t(15;19) observée dans les carcinomes de la ligne médiane (NMC), extrêmement agressifs et létaux. La protéine BRD4 possède un double bromodomaine capable de s'associer à la chromatine acétylée, et recrute divers facteurs sur la chromatine. NUT est une protéine de fonction inconnue exprimée exclusivement au cours de la spermatogenèse. J'ai pu démontrer que la protéine fusion BRD4-NUT était suffisante pour induire la tumorigenèse, par un mécanisme de séquestration de la proteine histone acétyltransférase (HAT) CBP/p300. NUT interagit avec CBP/p300 et suractive son activité d'acétylation, créant des foci hyperacétylés de chromatine. BRD4-NUT empêche ainsi CBP/p300 d'aller co-activer la transcription de nombreux gènes, et bloque notamment la réponse apoptotique p53-dépendante. Une inhibition de BRD4-NUT – par siRNA, mutation des bromodomaines ou dérégulation de l'acétylation des foci par des inhibiteurs des histones déacétylases (HDAC) – réenclenche la voie d'apoptose et la mort de ces cellules tumorales. Cette étude est un exemple précis de l'impact qu'une dérégulation épigénétique peut avoir sur l'homéostasie cellulaire et son mécanisme d'induction de la tumorigenèse. Je me suis également interessé à caracteriser la protéine NUT lors de son contexte physiologique, la spermatogenèse, ou lors de son expression illégitime dans des lignées tumorales sans fusion avec BRD4. La protéine NUT est exprimée au niveau des stades de maturation des cellules germinales spermatides, et pourrait participer au remodelage du génome et à l'établissement de l'épigénome final du spermatozoïde. Nut semble également conférer un avantage prolifératif lors de son expression anormale dans au moins trois lignées cellulaires, U2OS, A549 et A7R5. Ainsi, la protéine NUT, seule ou fusionnée avec BRD4, est un facteur Cancer/Testiculaire capable d'influer négativement sur l'homéostasie des cellules somatiques dans lesquelles ses fonctions, normalement restreintes à la spermatogenèse, participent à la tumorigenèse.

A descondensação da cromatina exerce um papel central nas diversas etapas do processo de carcinogênese abrindo o genoma para a ação de fatores de transcrição, exercendo papel na progressão e resistência tumoral. Bromodomínios e proteínas com terminal extra, como o BRD4, são leitores epigenéticos que regulam a expressão gênica e, portanto, também estão envolvidos na patogênese do câncer. O objetivo do presente estudo foi estudar o efeito da inibição do BRD4 no carcinoma espinocelular de cabeça e pescoço (CECP). Para esse propósito, foi utilizado JQ1, inibidor de BRD4, em concentração de 1uM, nas linhagens de carcinoma de cabeça e pescoço HN6, HN12 e HN13. Foi analisado os níveis de BRD4, H4 acetilada e SIRT1 fosforilado através de reações de imunofluorecência e p16ink4 por imunohistoquímica. Foi realizado western blot para checar os níveis de p53 e p53 acetilado. Ensaio de formação de colônias e câmera de invasão foram realizados para testar o efeito do inibidor na proliferação e invasão celular. Através da citometria de fluxo foi analisado o efeito da apoptose com a marcação de caspase-3 clivada, do ciclo celular através da reação por iodeto de propídio e ainda da população de células tronco tumorais pela análise de ALDH e CD44. Por fim, foi realizado modelo xenográfico subcutâneo para analisar o efeito do JQ1. Os resultados mostraram diminuição significativa da expressão de BRD4 e H4ac após tratamento com JQ1. As linhagens celulares mostraram redução na capacidade de invasão e de formação de colônias quando submetidas ao JQ1. Não foram encontradas diferenças em relação ao número de células caspase-3 clivada positivas. Por outro lado, foi encontrado um maior número de células na fase G1 do ciclo celular após o uso do inibidor estudado. As células tratadas com JQ1 mostraram menor expressão de p-SIRT1 o que levou a uma diminuição da acetilação do p53 e um aumento na expressão de p16ink4. Paralelamente, foi encontrado uma diminuição na população de células positivas para ALDH e CD44. Houve diminuição do crescimento do tumor no modelo xenográfico tratado com JQ1 quando comparado ao veículo. Nos tecidos derivados do ensaio in vivo, houve uma diminuição nos marcadores p16ink4, pSIRT1 além de acúmulo de H2AX. Conclui-se que o uso de JQ1 resulta na disrupção do crescimento do CECP associado a ativação de senescência, indução de dano de DNA além de reduzir a população de células tronco tumorais. Esses novos achados indicam que o BRD4 é um importante modificador epigenético nos CECP sendo um viável alvo terapêutico. / Chromatin descondensation plays a central step in the various stages of the carcinogenesis process opening the genome for transcription factors playing a role in tumor progress and resistance. Bromodomains and extra terminal family, as BRD4, are epigenetics readers that regulate gene expression thus they are also involved in cancer pathogenesis. The objective of this project was studied the effect of BRD4 in head and neck squamous cell carcinoma (HNSCC). For this purpose, JQ1, a BRD4 inhibitor, was used in 1uM concentration, in HN6, HN12 and HN13 head and neck carcinoma cell lines. The levels of BRD4, acetylates h4 and phosphorylated SIRT1 were analyzed by immunofluorescence and p16ink4 labeling by immunohistochemistry. Western blot was performed to check the levels of p53 and acetylated p53. Colony assay and invasion chamber were performed to test the inhibitory effect on cell proliferation and invasion. The effect of apoptosis with the cleaved caspase-3 labeling, the cell cycle by propidium iodide and of the population of tumor stem cells by the analysis of ALDH and CD44 was analyzed through flow cytometry. Finally, a subcutaneous xerographic model was performed to analyze the effect of JQ1. A significant decrease in the expression of BRD4 and H4ac was found after application of JQ1. The cell lines results showed a reduction in the capacity of invasion and also formation of colonies when submitted to JQ1. No differences were found in relation to the number of cells caspase-3 cleaved positives. On the other hand, a large number of cells were found in G1 arrest of cell cycle after use of the BRD4 inhibitor studied. Cells treated with JQ1 showed lower expression of p-SIRT1 which led to a decrease in p53 acetylation and an increase in p16ink4 expression. In parallel, a decrease of ALDH and CD44 positive cells population was found. A decrease in tumor growth was discovered when treated by JQ1 if compared to the vehicle. In tissues samples derived from the in vivo assay, there was a decrease in p16ink4, pSIRT1 markers in addition to -H2Ax accumulation. In conclusion JQ1 causes HNSSC tumor growth disruption associated a senescence activation, DNA damage and a reduce number of cancer stem cells. These new findings indicate that BRD4 is an important genetic modifier in HNSSC and is a viable therapeutic target.

A descondensação da cromatina exerce um papel central nas diversas etapas do processo de carcinogênese abrindo o genoma para a ação de fatores de transcrição, exercendo papel na progressão e resistência tumoral. Bromodomínios e proteínas com terminal extra, como o BRD4, são leitores epigenéticos que regulam a expressão gênica e, portanto, também estão envolvidos na patogênese do câncer. O objetivo do presente estudo foi estudar o efeito da inibição do BRD4 no carcinoma espinocelular de cabeça e pescoço (CECP). Para esse propósito, foi utilizado JQ1, inibidor de BRD4, em concentração de 1uM, nas linhagens de carcinoma de cabeça e pescoço HN6, HN12 e HN13. Foi analisado os níveis de BRD4, H4 acetilada e SIRT1 fosforilado através de reações de imunofluorecência e p16ink4 por imunohistoquímica. Foi realizado western blot para checar os níveis de p53 e p53 acetilado. Ensaio de formação de colônias e câmera de invasão foram realizados para testar o efeito do inibidor na proliferação e invasão celular. Através da citometria de fluxo foi analisado o efeito da apoptose com a marcação de caspase-3 clivada, do ciclo celular através da reação por iodeto de propídio e ainda da população de células tronco tumorais pela análise de ALDH e CD44. Por fim, foi realizado modelo xenográfico subcutâneo para analisar o efeito do JQ1. Os resultados mostraram diminuição significativa da expressão de BRD4 e H4ac após tratamento com JQ1. As linhagens celulares mostraram redução na capacidade de invasão e de formação de colônias quando submetidas ao JQ1. Não foram encontradas diferenças em relação ao número de células caspase-3 clivada positivas. Por outro lado, foi encontrado um maior número de células na fase G1 do ciclo celular após o uso do inibidor estudado. As células tratadas com JQ1 mostraram menor expressão de p-SIRT1 o que levou a uma diminuição da acetilação do p53 e um aumento na expressão de p16ink4. Paralelamente, foi encontrado uma diminuição na população de células positivas para ALDH e CD44. Houve diminuição do crescimento do tumor no modelo xenográfico tratado com JQ1 quando comparado ao veículo. Nos tecidos derivados do ensaio in vivo, houve uma diminuição nos marcadores p16ink4, pSIRT1 além de acúmulo de H2AX. Conclui-se que o uso de JQ1 resulta na disrupção do crescimento do CECP associado a ativação de senescência, indução de dano de DNA além de reduzir a população de células tronco tumorais. Esses novos achados indicam que o BRD4 é um importante modificador epigenético nos CECP sendo um viável alvo terapêutico. / Chromatin descondensation plays a central step in the various stages of the carcinogenesis process opening the genome for transcription factors playing a role in tumor progress and resistance. Bromodomains and extra terminal family, as BRD4, are epigenetics readers that regulate gene expression thus they are also involved in cancer pathogenesis. The objective of this project was studied the effect of BRD4 in head and neck squamous cell carcinoma (HNSCC). For this purpose, JQ1, a BRD4 inhibitor, was used in 1uM concentration, in HN6, HN12 and HN13 head and neck carcinoma cell lines. The levels of BRD4, acetylates h4 and phosphorylated SIRT1 were analyzed by immunofluorescence and p16ink4 labeling by immunohistochemistry. Western blot was performed to check the levels of p53 and acetylated p53. Colony assay and invasion chamber were performed to test the inhibitory effect on cell proliferation and invasion. The effect of apoptosis with the cleaved caspase-3 labeling, the cell cycle by propidium iodide and of the population of tumor stem cells by the analysis of ALDH and CD44 was analyzed through flow cytometry. Finally, a subcutaneous xerographic model was performed to analyze the effect of JQ1. A significant decrease in the expression of BRD4 and H4ac was found after application of JQ1. The cell lines results showed a reduction in the capacity of invasion and also formation of colonies when submitted to JQ1. No differences were found in relation to the number of cells caspase-3 cleaved positives. On the other hand, a large number of cells were found in G1 arrest of cell cycle after use of the BRD4 inhibitor studied. Cells treated with JQ1 showed lower expression of p-SIRT1 which led to a decrease in p53 acetylation and an increase in p16ink4 expression. In parallel, a decrease of ALDH and CD44 positive cells population was found. A decrease in tumor growth was discovered when treated by JQ1 if compared to the vehicle. In tissues samples derived from the in vivo assay, there was a decrease in p16ink4, pSIRT1 markers in addition to -H2Ax accumulation. In conclusion JQ1 causes HNSSC tumor growth disruption associated a senescence activation, DNA damage and a reduce number of cancer stem cells. These new findings indicate that BRD4 is an important genetic modifier in HNSSC and is a viable therapeutic target.

A descondensação da cromatina exerce um papel central nas diversas etapas do processo de carcinogênese abrindo o genoma para a ação de fatores de transcrição, exercendo papel na progressão e resistência tumoral. Bromodomínios e proteínas com terminal extra, como o BRD4, são leitores epigenéticos que regulam a expressão gênica e, portanto, também estão envolvidos na patogênese do câncer. O objetivo do presente estudo foi estudar o efeito da inibição do BRD4 no carcinoma espinocelular de cabeça e pescoço (CECP). Para esse propósito, foi utilizado JQ1, inibidor de BRD4, em concentração de 1uM, nas linhagens de carcinoma de cabeça e pescoço HN6, HN12 e HN13. Foi analisado os níveis de BRD4, H4 acetilada e SIRT1 fosforilado através de reações de imunofluorecência e p16ink4 por imunohistoquímica. Foi realizado western blot para checar os níveis de p53 e p53 acetilado. Ensaio de formação de colônias e câmera de invasão foram realizados para testar o efeito do inibidor na proliferação e invasão celular. Através da citometria de fluxo foi analisado o efeito da apoptose com a marcação de caspase-3 clivada, do ciclo celular através da reação por iodeto de propídio e ainda da população de células tronco tumorais pela análise de ALDH e CD44. Por fim, foi realizado modelo xenográfico subcutâneo para analisar o efeito do JQ1. Os resultados mostraram diminuição significativa da expressão de BRD4 e H4ac após tratamento com JQ1. As linhagens celulares mostraram redução na capacidade de invasão e de formação de colônias quando submetidas ao JQ1. Não foram encontradas diferenças em relação ao número de células caspase-3 clivada positivas. Por outro lado, foi encontrado um maior número de células na fase G1 do ciclo celular após o uso do inibidor estudado. As células tratadas com JQ1 mostraram menor expressão de p-SIRT1 o que levou a uma diminuição da acetilação do p53 e um aumento na expressão de p16ink4. Paralelamente, foi encontrado uma diminuição na população de células positivas para ALDH e CD44. Houve diminuição do crescimento do tumor no modelo xenográfico tratado com JQ1 quando comparado ao veículo. Nos tecidos derivados do ensaio in vivo, houve uma diminuição nos marcadores p16ink4, pSIRT1 além de acúmulo de H2AX. Conclui-se que o uso de JQ1 resulta na disrupção do crescimento do CECP associado a ativação de senescência, indução de dano de DNA além de reduzir a população de células tronco tumorais. Esses novos achados indicam que o BRD4 é um importante modificador epigenético nos CECP sendo um viável alvo terapêutico. / Chromatin descondensation plays a central step in the various stages of the carcinogenesis process opening the genome for transcription factors playing a role in tumor progress and resistance. Bromodomains and extra terminal family, as BRD4, are epigenetics readers that regulate gene expression thus they are also involved in cancer pathogenesis. The objective of this project was studied the effect of BRD4 in head and neck squamous cell carcinoma (HNSCC). For this purpose, JQ1, a BRD4 inhibitor, was used in 1uM concentration, in HN6, HN12 and HN13 head and neck carcinoma cell lines. The levels of BRD4, acetylates h4 and phosphorylated SIRT1 were analyzed by immunofluorescence and p16ink4 labeling by immunohistochemistry. Western blot was performed to check the levels of p53 and acetylated p53. Colony assay and invasion chamber were performed to test the inhibitory effect on cell proliferation and invasion. The effect of apoptosis with the cleaved caspase-3 labeling, the cell cycle by propidium iodide and of the population of tumor stem cells by the analysis of ALDH and CD44 was analyzed through flow cytometry. Finally, a subcutaneous xerographic model was performed to analyze the effect of JQ1. A significant decrease in the expression of BRD4 and H4ac was found after application of JQ1. The cell lines results showed a reduction in the capacity of invasion and also formation of colonies when submitted to JQ1. No differences were found in relation to the number of cells caspase-3 cleaved positives. On the other hand, a large number of cells were found in G1 arrest of cell cycle after use of the BRD4 inhibitor studied. Cells treated with JQ1 showed lower expression of p-SIRT1 which led to a decrease in p53 acetylation and an increase in p16ink4 expression. In parallel, a decrease of ALDH and CD44 positive cells population was found. A decrease in tumor growth was discovered when treated by JQ1 if compared to the vehicle. In tissues samples derived from the in vivo assay, there was a decrease in p16ink4, pSIRT1 markers in addition to -H2Ax accumulation. In conclusion JQ1 causes HNSSC tumor growth disruption associated a senescence activation, DNA damage and a reduce number of cancer stem cells. These new findings indicate that BRD4 is an important genetic modifier in HNSSC and is a viable therapeutic target.

The inevitable progression of advanced prostate cancer to castration resistance, and ultimately to lethal metastatic disease, depends on primary or acquired resistance to conventional androgen-deprivation therapy (ADT) and accumulated resistance mechanisms to evade androgen receptor (AR) suppression. Whereas the canonical androgen/AR signaling axis maintains prostate cell growth, differentiation and survival, in prostate cancer cells, AR adaptations that arise in response to ADT are not singular, but diverse, and include gene amplification, mutation and even complete loss of receptor expression. Collectively, each of these AR adaptations contributes to a complex, heterogenous, ADT-resistant tumor that culminates in prostate tumor cells transitioning from epithelial to mesenchymal states (EMT) and the development of metastatic castration-resistant prostate cancer (mCRPC). Here, we examined prostate cancer cell lines that model common CRPC subtypes, each with different AR composition, and focused on novel regulators of tumor progression, the Bromodomain and ExtraTerminal (BET – BRD2, BRD3 and BRD4) family of proteins, to test the hypothesis that each BET family member regulates EMT and underlying characteristics such as cell motility and invasiveness. We systematically manipulated the BET proteins and found that BRD4 regulates cell migration and invasion across all models of CRPC, regardless of aggressiveness and AR status, whereas BRD2 and BRD3 only regulate cell migration and invasion in less aggressive models that retain AR expression or signaling. We determined that BRD4’s contribution to this process occurs through the transcriptional regulation of AHNAK, SNAI1 and SNAI2, which are EMT genes linked to promotion of metastasis in a diverse set of cancers. Furthermore, treatment of CRPC cell lines with low doses of MZ1, a small-molecule, BRD4-selective degrader, inhibits EMT and metastatic potential. Overall, these results reveal a novel, BRD4-regulated EMT gene signature that may be targetable to treat metastatic castration-resistant prostate cancer.

L’ oncoprotéine E6 - qui joue un rôle crucial dans le processus d’oncogenèse induit par les papillomavirus a longtemps résisté à toute analyse. Depuis 1995 l’équipe Oncoprotéines a concentré ses efforts sur cette problématique. Ce qui a permis la résolution par RMN de la structure du domaine C-terminal de E6 en 2006. C’est dans ce cadre que j’ai commencé ce Doctorat en 2008, avec objectif de continuer la quête de données structurales sur E6 tout en acquérant des informations sur ses modes d’interaction avec ses cibles cellulaires. Les travaux de cette thèse ont permis l’obtention de la structure cristallographique de E6 (HPV16) en complexe avec un peptide de E6AP, en utilisant une approche originale capable de produire des protéines E6 stables et solubles. Cette structure constitue la première information structurale publiée sur des protéines E6 entières, attendue depuis plus de 20 ans par la communauté scientifique. J’ai effectué également durant cette thèse une analyse du système d’interaction de la protéine E6 basée sur une large étude d’interaction entre les protéines E6 (7 types) et 93 peptides porteurs de motif LxxLL. / The oncoprotein E6 which plays a crucial role in the process of carcinogenesis induced by HPV, has withstood all tests for a long time. Since 1995 the team « oncoproteins » has focused on this issue. This allowed the resolution of structure of C-terminal domain of E6 by NMR analysis, in 2006. In this context, i started my PhD in 2008 with aim to continue the pursuit of structural data on E6 while also acquiring information about its modes of interaction with its cellular targets. The work of this thesis has enabled us to obtain the crystal structure of E6 (HPV16) in complex with a peptide of E6AP, using an original approach capable of producing stable and soluble proteins E6. This structure represents the first structural information on full-length E6, awaited for over 20 years by the scientific community. I also performed during this thesis an analysis of the interaction system of the E6 protein based on a large study of interaction between proteins E6 (7 types) and 93 peptides bearing LxxLL motif.