Spelling suggestions: "subject:"aurora kinase""
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Evaluation de l'activité anti-tumorale de thérapeutiques ciblées dans les sarcomes : implication des Aurora kinases et de CHK1 / Assessment of anti-tumoral activity of targeted therapies in sarcomas : Aurora kinases and CHK1Mattei, Jean-Camille 16 December 2016 (has links)
Les sarcomes sont des cancers rares touchant toutes les zones du corps humain, caractérisés par une grande diversité de nature, de comportement clinique et de réponse aux thérapies existantes, certains étant de bon pronostic, d’autres très difficilement curables.Leur traitement de référence est la chirurgie ; la radiothérapie et les protocoles de chimiothérapie n’ayant que peu évolué lors des 30 dernières années.Récemment des caractéristiques génétiques leur étant propres ont été découvertes, prédictives de leur agressivité et contre lesquelles il est possible de diriger des drogues spécifiques pouvant améliorer le pronostic et diminuer les effets secondaires des thérapies conventionnelles.C’est sur l’inhibition d’Aurora Kinase A et B et CHK1 que s’est focalisé ce travail avec le test des effets de deux nouvelles drogues sur 9 types de cellules cancéreuses sarcomateuses avec des résultats très prometteurs, qu’il conviendra de conforter par d’autres expériences, notamment sur l’animal. / Sarcomas are rare cancers, which may arise in all parts of human body. They are characterized by great diversity in their nature, clinical behavior and response to existing therapeutics. Some are of good prognosis and others hard to cure.Their treatment essentially relies on surgery and radiotherapy or chemotherapy haven’t know major breakthrough over the last 3 decades.Recently new genetics abnormalities linked to sarcomas have been discovered. Their analysis can predict their aggressiveness and it is now possible to develop targeted therapies against them. This could help improving cancer prognosis and/or limiting conventional drugs adverse effects.Our work focused on Aurora Kinase A and B and CHK1 inhibition, testing the effects of 2 new drugs on 9 types of sarcoma cells with promising results, which we will confort by other experiments, including on the animal.
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Mecanismos Envolvidos com a Inibição de Aurora-Quinases em Carcinoma de Adrenal / Mechanisms Involved in the Inhibition of Aurora Kinases in Adrenal CarcinomaBorges, Kleiton Silva 09 May 2014 (has links)
Introdução: Tumores adrenocorticais (TAC) são raros, correspondendo somente a 0,2% de todas as neoplasias pediátricas, sendo que a maioria dos casos são diagnosticados no Brasil e estão associados com a mutação TP53 p.R337H. A cirurgia é o único tratamento efetivo conhecido para os TAC, sendo os tumores em estadios avançados frequentemente fatais. A família das Aurora-quinases é formada por três membros (Aurora-A, -B e -C) os quais atuam em diversas fases do ciclo celular, como alinhamento dos cromossomos, formação do fuso mitótico e citocinese. Diferentes trabalhos mostraram a expressão alterada de membros desta família em vários tipos de tumores e a inibição da atividade destas proteínas tem sido considerada uma potencial abordagem para o tratamento do câncer. Objetivo: A partir da análise da expressão dos genes Aurora-A e Aurora-B em amostras de TAC pediátrico, foram investigados os efeitos do AMG 900, um pan-inibidor de aurora quinases, na proliferação, apoptose, síntese hormonal e perfil transcricional da linhagem H295A. Além disso, foram avaliados os efeitos do AMG 900 combinado com diferentes quimioterápicos. Metodologia: Os níveis de expressão dos genes Aurora-A e Aurora-B foram analisados em 60 crianças com TAC através das técnicas de RT-qPCR e imuno-histoquímica. A proliferação celular foi avaliada por coloração com Giemsa e a apoptose foi realizada por citometria de fluxo. A análise de combinação de drogas foi feita com base no método de Chou-Talalay e o ensaio de microarray foi realizado utilizando a plataforma da Agilent. Resultados: A expressão dos genes Aurora-A e Aurora-B foi associada com estadios avançados da doença e a expressão do Aurora-A foi associada com a presença da mutação TP53 p.R337H. O tratamento com o AMG900 causou a inibição da proliferação, aumento da apoptose e sensibilizou as células para os inibidores de topoisomerase II (doxorrubicina e etoposídeo). Adicionalmente, o AMG 900 levou à redução da síntese de hormônios bem como modulou a expressão de genes envolvidos com esta atividade. A inibição das aurora-quinases alterou a expressão de genes associados com a regulação da fase G1 do ciclo celular e afetou a expressão de genes da via de sinalização Notch. Conclusão: A inibição das aurora-quinases pelo AMG 900 pode ser uma alternativa para o tratamento dos tumores adrenocorticais. / Introduction: Pediatric adrenocortical tumors (ACT) are rare malignancies representing only 0.2 % of all pediatric cancers. Most cases are diagnosed in Brazil and are associated with TP53 p.R337H mutation. Surgery is the only effective treatment known for the ACT however this approach has a small impact on survival in advanced disease. The Aurora kinase family is comprised of three members (Aurora-A, -B and -C) which act at different phases of the cell cycle, such as chromosomes alignment, mitotic spindle formation and cytokinesis. Several studies have demonstrated altered expression of members of this family in various types of tumors and the functional inhibition of the aurora kinases have been considered as a potential approach to cancer treatment. Aim: On the basis of analysis of Aurora-A and Aurora-B gene expression in the samples from pediatric ACT, we investigated the effects of AMG 900, a pan-aurora kinase inhibitor, on proliferation, apoptosis rate, hormone synthesis and transcriptional profile of H295A cell line. Furthermore, we evaluated the effects of AMG 900 combined with different chemotherapeutic agents. Methods: The mRNA expression levels of Aurora-A and Aurora-B genes were analyzed in 60 children with ACT by RT-qPCR and immunohistochemistry. Cell proliferation was assessed by Giemsa staining and apoptosis was performed by flow cytometry. Drug combination analysis was made on the basis of Chou- Talalay method. Microarray experiments were carried out using the Agilent human microarray. Results: Aurora-A and Aurora-B overexpression was associated with advanced disease. Patients carrying the TP53 p.R337H mutation presented significantly higher expression values of Aurora-A. Treatment with AMG900 caused inhibition of proliferation, increased apoptosis and sensitized the cells to topoisomerase II inhibitors (doxorubicin and etoposide). Additionally, the AMG 900 led to decreased synthesis of hormones and modulated the expression of genes involved in this activity. Finally, Aurora kinases inhibition altered the expression of genes associated with G1 cell cycle phase regulation and affected the Notch signaling pathway target genes. Conclusion: These data suggest that Aurora kinase inhibition by AMG900 may be a new therapeutic approach to adrenocortical carcinoma treatment.
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Investigação das quinases Aurora A e Aurora B como potenciais alvos terapêuticos no câncer de pulmão induzido pelo oncogene KRAS / Investigation of Aurora A and Aurora B kinases as potential targets in KRAS-induced lung cancerSantos, Edmilson Ozorio dos 27 November 2013 (has links)
As alterações genéticas mais frequentes em tumores de pulmão são mutações pontuais que ativam o oncogene KRAS. Apesar destas mutações estarem ligadas à oncogênese de forma causal, diferentes abordagens para inibir as proteínas RAS diretamente fracassaram na clínica. Portanto, para que melhores alvos terapêuticos para o câncer de pulmão se tornem disponíveis, será necessário identificar as vias sinalizadoras ativadas pela proteína KRAS, que são críticas para a oncogênese. O objetivo deste projeto foi identificar novos alvos terapêuticos na oncogênese pulmonar induzida pela KRAS. Este projeto se baseou na seguinte hipótese: (1) a KRAS oncogênica leva à ativação das quinases mitóticas Aurora A e/ou B e (2) que as quinases Aurora A e/ou B são alvos terapêuticos relevantes no câncer de pulmão induzido pelo oncogene KRAS. Esta hipótese foi formulada com base em estudos anteriores mostrando que a quinase Aurora A fosforila diretamente componentes das vias efetoras de RAS, e que a Aurora A e Aurora B cooperam com a RAS oncogênica na transformação maligna. Para testar esta hipótese, nós inicialmente determinamos se a forma oncogênica da KRAS induz a expressão das quinases Aurora A e B. Para tanto, nós usamos 3 modelos celulares: (1) uma linhagem primária epitelial pulmonar imortalizada e seu par isogênico transformado pela KRAS oncogênica; (2) células tumorais pulmonares H1703 manipuladas geneticamente para expressar a forma oncogênica da KRAS de forma induzível; e (3) células de adenocarcinoma pulmonar portadoras de mutações oncogênicas em KRAS H358 e A549 manipuladas geneticamente para expressar short hairpin RNAs (shRNAs) para KRAS de forma induzível. Em todos os casos, a expressão da forma oncogênica da KRAS se correlacionou positivamente com a expressão de Aurora A e B. Para validar as quinases Aurora A e B como alvos relevantes do ponto de vista terapêutico, nós usamos, nas células mencionadas acima, abordagens genéticas ou farmacológicas para inibir a expressão ou atividade das quinases Aurora A e B. Nas células A549 e H358, portadoras da forma oncogênica da KRAS, a inibição da expressão das quinases Aurora A ou B por interferência de RNA de forma induzível, bem como o tratamento com um inibidor dual destas quinases, reduziu o crescimento, viabilidade e tumorigenicidade celulares in vitro. Mais importante do que isso, no modelo celular primário isogênico, bem como na linhagem H1703 com expressão induzível de KRAS oncogênica, a inibição farmacológica dual das quinases Aurora A e B levou a uma redução no crescimento, viabilidade e tumorigenicidade celulares de forma dependente da presença da KRAS oncogênica, sugerindo que a inibição das quinases Aurora A e B afeta especificamente células transformadas pela KRAS. Em conclusão, nossos resultados apoiam a nossa hipótese de que as quinases Aurora são alvos da KRAS oncogênica no pulmão, e sugerem a inibição das quinases Aurora como uma nova abordagem para a terapia do câncer de pulmão induzido pela forma oncogênica da KRAS. / The most frequent genetic change found in lung tumors are activating point mutations in the KRAS gene, which have been causally linked to the oncogenic process. Unfortunately, different approaches to target RAS proteins for therapy have been unsuccessful. Therefore, in order to select better targets for lung cancer therapy, key cancer-relevant KRAS downstream pathways will need to be identified. The overall objective of this study was to identify novel therapeutic targets in KRAS-mediated lung cancer. This project was based on the following hypothesis: (1) KRAS activates mitotic kinases Aurora A and/or B; and (2) Aurora A and/or B are relevant therapeutic targets in KRAS-induced lung cancer. This hypothesis was formulated on the basis of published studies showing that Aurora A directly phosphorylates RAS effector pathway components, and Aurora A and B both cooperate with oncogenic RAS to promote malignant transformation. In order to test this hypothesis, we first determined whether oncogenic KRAS induces Aurora kinase expression. For that purpose, we used three different cell-based models: (1) an immortalized primary lung epithelial cell line and its isogenic KRAS-transformed counterpart, (2) H1703 lung cancer cell line engineered to express oncogenic KRAS inducibly, and (3) KRAS positive lung cancer cell lines H358 and A549 stably expressing inducible shRNAs targeting KRAS. In all cases, KRAS expression positively correlated with Aurora A and Aurora B expression. In order to validate Aurora A and/or B as therapeutically relevant KRAS targets in lung cancer, we used genetic and/or pharmacological approaches in the abovementioned cells to inactivate Aurora A or B. In KRAS positive H358 and A549 cell lines, inducible shRNA-mediated knockdown of Aurora A or B, as well as treatment with a dual Aurora A and B inhibitor, decreased growth, viability and tumorigenicity in vitro. More importantly, in the primary isogenic model and in the H1703 KRAS-inducible cell line, dual pharmacological inhibiton of Aurora A and B reduced growth, viability and tumorigenicity in an oncogenic KRAS-dependent manner. This suggests that Aurora kinase inhibition therapy can specifically target KRAS transformed cells. In conclusion, our results support our hypothesis that Aurora kinases are important KRAS targets in lung cancer and suggest Aurora kinase inhibition as a novel approach for KRAS-induced lung cancer therapy.
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Estudo da modulação da via Wnt pelo inibidor de Aurora-quinases AMG900 em linhagens celulares de meduloblastoma pediátrico / Study of Modulation of the Wnt pathway by Aurora kinases inhibitor AMG900 in pediatric medulloblastoma cell linesGeron, Lenisa 12 January 2016 (has links)
O meduloblastoma (MB) é o tumor cerebral maligno mais comum na infância. A formação/progressão desta neoplasia foi associada a alterações moleculares, que inclui a desregulação da via de sinalização Wingless (Wnt), responsável pelo desenvolvimento embrionário. Além disso, as proteínas da família Aurora-quinases (A, B e C) têm sido amplamente estudadas, uma vez que a Aurora A e B foram encontrados hiperexpressas em diversas neoplasias, como o MB. Estudos recentes mostraram que existe uma associação entre a Via Wnt e as Aurora-quinases. No entanto, poucos trabalhos foram realizados para confirmar essa associação. Ademais, não existem trabalhos que relatem os efeitos do AMG900, um pan-inibidor de aurora-quinases, em MB, dando enfoque na regulação da via Wnt. Assim, o objetivo deste trabalho foi avaliar a modulação da via Wnt pelo inibidor AMG900 nas linhagens celulares de meduloblastoma pediátrico. Foram realizados os ensaios de PCR convencional, sequenciamento, qRT-PCR, transfecção transiente, ensaio clonogênico, Western Blot e ciclo celular. As linhagens celulares UW402, UW473 e ONS-76 não apresentaram mutações no éxon 3 do gene CTNNB1 (?-catenina) e no éxon 15 do gene APC. Não foi observada uma expressão significativa de CTNNB1, confirmando que as linhagens não possuíam a via Wnt ativa. Com isso foi necessário a transfecção transiente com a ?- catenina. Após este ensaio, houve um aumento da expressão de CTNNB1, Ciclina D1 e CMyc nas três linhagens, o que não ocorreu com as Auroras A e B. No ensaio clonogênico foi observado uma redução do número de colônias nas linhagens UW473 e ONS-76. Observou-se um aumento da expressão proteica da ?-catenina, da Aurora A e B na UW473, o que ocorreu somente com a ?-catenina na linhagem ONS-76. Após o tratamento com o AMG900 ocorreu uma diminuição da expressão proteica de ?-catenina, da Aurora A e B em ambas as linhagens. A transfecção não alterou o percentil celular em G2/M na UW402 e UW473. Já na ONS-76 houve um aumento significativo em G2/M, e o AMG900 potencializou esse bloqueio apenas nessa linhagem. Os resultados sugerem que pode haver alguma relação entre a inibição das proteínas Aurora-quinases e a expressão de proteínas da via Wnt. / Medulloblastoma (MB) is the most common malignant brain tumor in childhood. Tumor formation/progression has been associated to molecular alterations that include dysregulation of signaling pathway Wingless (Wnt), responsible for embryonic development. In addition, cell cycle proteins Aurora-kinase (A, B and C) have been widely studied since Aurora A and B were found overexpressed in many cancers such as MB. Recent studies show that there is an association between Wnt pathway and Aurora kinase proteins. However, few studies have been conducted to confirm this association. Moreover, there are no studies reporting the effects of AMG900 in MB, by focusing on the regulation of the Wnt pathway. The aim of this study is to evaluate Wnt pathway modulation by Aurora kinases inhibitor AMG900 in pediatric medulloblastoma cell lines. Conventional PCR, sequencing, qRT-PCR, transient transfection, clonogenic assay, Western Blot and cell cycle assays were performed. UW402, UW473 and ONS-76 cell lines did not present mutations in exon 3 of CTNNB1 gene and exon 15 of APC gene. There was no significant expression of CTNNB1 and their target genes in these cell lines, confirming that they did not have Wnt pathway activated. Considering this, transient transfection was necessary. After this trial, there was an increase in expression of CTNNB1 gene and its target genes Cyclin D1 and C-Myc in the three cell lines, which was not observed in Aurora kinases. Furthermore, in the clonogenic assay, a reduction in the number of colonies in UW473 and ONS-76 cell lines was observed. It was also observed an increase in ?-catenin protein, Aurora A and B in UW473 cell line, but not in ONS-76 cell line. However, after treatment there was a decrease in protein expression of ?-catenin, Aurora A and B in both cells. Transfection did not change the cellular percentile in G2 / M in UW402 and UW473. In ONS-76 there was a significant increase in G2 / M, and the treatment with AMG900 potentiated this block only in this cell line. Results suggest that there may be some relation between the inhibition of Aurora kinase protein and protein expression in Wnt pathway.
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Myc-induced Lymphomagenesis : In vivo assessment of downstream pathways / Myc-inducerad lymfomutveckling : Utvärdering av målgener in vivoRimpi, Sara January 2010 (has links)
Myc oncogenes encode transcription factors that bind to E-box sequences in DNA, driving the expression of a large number of target genes and are deregulated in approximately 70% of human cancers. Deregulated Myc expression cause enhanced proliferation (which is counteracted by apoptosis), angiogenesis and cancer. Though Myc’s importance in induction of S phase has been established, less is known about its functions in the G2 and M phases of the cell cycle. Paper I addresses the targeting of the Myc targets Aurora kinase A and B that have roles in G2/M transition and provide evidence that pharmaceutical Aurora kinase inhibition causes cell cycle arrest and apoptosis in a Myc-selective manner and is useful in treating Myc-induced lymphomas in vivo. The assumption that the important target genes responsible for the biological effects of Myc overexpression were those encoding components of the cell cycle machinery lead to little interest in other potentially important groups of target genes. However, recent work challenged this view by indicating that Myc target genes encoding metabolic enzymes may be critical for Myc-induced tumorigenesis. Importantly, the targeting of Myc target genes encoding metabolic enzymes has the potential of providing a new treatment strategy of Myc-induced cancers. Paper II covers the pharmaceutical targeting of the Myc-induced spermidine synthase (Srm) that shows promise as a tool for chemoprevention by affecting proliferation, but not for the treatment of established tumors. Paper III focuses on the negligible effect an Ldha mutation has on Myc- induced lymphomagenesis. Ldha has long been known to be a Myc target gene and in vitro experiments have recently indicated it to be important for transformation. It seems the negligible effect of the Ldh mutation can be explained by the high frequency of loss of either Arf or p53 in this mouse model, since enforced Ras-Myc oncogenic cooperation in soft agar assays of Ldh mutant MEFs effectively inhibits colony formation, and λ-Myc;Ldh mutant bone marrow infected with oncogenic Ras does not give rise to tumors when transplanted into wild-type mice. A role for Ldh in the ability of tumors to evade the immune system was also indicated in this study. The combined experiences and very different outcome of the three studies included in this thesis draw attention to the value of in vivo assessment of Myc downstream targets in Myc-induced lymphomagenesis.
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Investigação das quinases Aurora A e Aurora B como potenciais alvos terapêuticos no câncer de pulmão induzido pelo oncogene KRAS / Investigation of Aurora A and Aurora B kinases as potential targets in KRAS-induced lung cancerEdmilson Ozorio dos Santos 27 November 2013 (has links)
As alterações genéticas mais frequentes em tumores de pulmão são mutações pontuais que ativam o oncogene KRAS. Apesar destas mutações estarem ligadas à oncogênese de forma causal, diferentes abordagens para inibir as proteínas RAS diretamente fracassaram na clínica. Portanto, para que melhores alvos terapêuticos para o câncer de pulmão se tornem disponíveis, será necessário identificar as vias sinalizadoras ativadas pela proteína KRAS, que são críticas para a oncogênese. O objetivo deste projeto foi identificar novos alvos terapêuticos na oncogênese pulmonar induzida pela KRAS. Este projeto se baseou na seguinte hipótese: (1) a KRAS oncogênica leva à ativação das quinases mitóticas Aurora A e/ou B e (2) que as quinases Aurora A e/ou B são alvos terapêuticos relevantes no câncer de pulmão induzido pelo oncogene KRAS. Esta hipótese foi formulada com base em estudos anteriores mostrando que a quinase Aurora A fosforila diretamente componentes das vias efetoras de RAS, e que a Aurora A e Aurora B cooperam com a RAS oncogênica na transformação maligna. Para testar esta hipótese, nós inicialmente determinamos se a forma oncogênica da KRAS induz a expressão das quinases Aurora A e B. Para tanto, nós usamos 3 modelos celulares: (1) uma linhagem primária epitelial pulmonar imortalizada e seu par isogênico transformado pela KRAS oncogênica; (2) células tumorais pulmonares H1703 manipuladas geneticamente para expressar a forma oncogênica da KRAS de forma induzível; e (3) células de adenocarcinoma pulmonar portadoras de mutações oncogênicas em KRAS H358 e A549 manipuladas geneticamente para expressar short hairpin RNAs (shRNAs) para KRAS de forma induzível. Em todos os casos, a expressão da forma oncogênica da KRAS se correlacionou positivamente com a expressão de Aurora A e B. Para validar as quinases Aurora A e B como alvos relevantes do ponto de vista terapêutico, nós usamos, nas células mencionadas acima, abordagens genéticas ou farmacológicas para inibir a expressão ou atividade das quinases Aurora A e B. Nas células A549 e H358, portadoras da forma oncogênica da KRAS, a inibição da expressão das quinases Aurora A ou B por interferência de RNA de forma induzível, bem como o tratamento com um inibidor dual destas quinases, reduziu o crescimento, viabilidade e tumorigenicidade celulares in vitro. Mais importante do que isso, no modelo celular primário isogênico, bem como na linhagem H1703 com expressão induzível de KRAS oncogênica, a inibição farmacológica dual das quinases Aurora A e B levou a uma redução no crescimento, viabilidade e tumorigenicidade celulares de forma dependente da presença da KRAS oncogênica, sugerindo que a inibição das quinases Aurora A e B afeta especificamente células transformadas pela KRAS. Em conclusão, nossos resultados apoiam a nossa hipótese de que as quinases Aurora são alvos da KRAS oncogênica no pulmão, e sugerem a inibição das quinases Aurora como uma nova abordagem para a terapia do câncer de pulmão induzido pela forma oncogênica da KRAS. / The most frequent genetic change found in lung tumors are activating point mutations in the KRAS gene, which have been causally linked to the oncogenic process. Unfortunately, different approaches to target RAS proteins for therapy have been unsuccessful. Therefore, in order to select better targets for lung cancer therapy, key cancer-relevant KRAS downstream pathways will need to be identified. The overall objective of this study was to identify novel therapeutic targets in KRAS-mediated lung cancer. This project was based on the following hypothesis: (1) KRAS activates mitotic kinases Aurora A and/or B; and (2) Aurora A and/or B are relevant therapeutic targets in KRAS-induced lung cancer. This hypothesis was formulated on the basis of published studies showing that Aurora A directly phosphorylates RAS effector pathway components, and Aurora A and B both cooperate with oncogenic RAS to promote malignant transformation. In order to test this hypothesis, we first determined whether oncogenic KRAS induces Aurora kinase expression. For that purpose, we used three different cell-based models: (1) an immortalized primary lung epithelial cell line and its isogenic KRAS-transformed counterpart, (2) H1703 lung cancer cell line engineered to express oncogenic KRAS inducibly, and (3) KRAS positive lung cancer cell lines H358 and A549 stably expressing inducible shRNAs targeting KRAS. In all cases, KRAS expression positively correlated with Aurora A and Aurora B expression. In order to validate Aurora A and/or B as therapeutically relevant KRAS targets in lung cancer, we used genetic and/or pharmacological approaches in the abovementioned cells to inactivate Aurora A or B. In KRAS positive H358 and A549 cell lines, inducible shRNA-mediated knockdown of Aurora A or B, as well as treatment with a dual Aurora A and B inhibitor, decreased growth, viability and tumorigenicity in vitro. More importantly, in the primary isogenic model and in the H1703 KRAS-inducible cell line, dual pharmacological inhibiton of Aurora A and B reduced growth, viability and tumorigenicity in an oncogenic KRAS-dependent manner. This suggests that Aurora kinase inhibition therapy can specifically target KRAS transformed cells. In conclusion, our results support our hypothesis that Aurora kinases are important KRAS targets in lung cancer and suggest Aurora kinase inhibition as a novel approach for KRAS-induced lung cancer therapy.
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Estudo da modulação da via Wnt pelo inibidor de Aurora-quinases AMG900 em linhagens celulares de meduloblastoma pediátrico / Study of Modulation of the Wnt pathway by Aurora kinases inhibitor AMG900 in pediatric medulloblastoma cell linesLenisa Geron 12 January 2016 (has links)
O meduloblastoma (MB) é o tumor cerebral maligno mais comum na infância. A formação/progressão desta neoplasia foi associada a alterações moleculares, que inclui a desregulação da via de sinalização Wingless (Wnt), responsável pelo desenvolvimento embrionário. Além disso, as proteínas da família Aurora-quinases (A, B e C) têm sido amplamente estudadas, uma vez que a Aurora A e B foram encontrados hiperexpressas em diversas neoplasias, como o MB. Estudos recentes mostraram que existe uma associação entre a Via Wnt e as Aurora-quinases. No entanto, poucos trabalhos foram realizados para confirmar essa associação. Ademais, não existem trabalhos que relatem os efeitos do AMG900, um pan-inibidor de aurora-quinases, em MB, dando enfoque na regulação da via Wnt. Assim, o objetivo deste trabalho foi avaliar a modulação da via Wnt pelo inibidor AMG900 nas linhagens celulares de meduloblastoma pediátrico. Foram realizados os ensaios de PCR convencional, sequenciamento, qRT-PCR, transfecção transiente, ensaio clonogênico, Western Blot e ciclo celular. As linhagens celulares UW402, UW473 e ONS-76 não apresentaram mutações no éxon 3 do gene CTNNB1 (?-catenina) e no éxon 15 do gene APC. Não foi observada uma expressão significativa de CTNNB1, confirmando que as linhagens não possuíam a via Wnt ativa. Com isso foi necessário a transfecção transiente com a ?- catenina. Após este ensaio, houve um aumento da expressão de CTNNB1, Ciclina D1 e CMyc nas três linhagens, o que não ocorreu com as Auroras A e B. No ensaio clonogênico foi observado uma redução do número de colônias nas linhagens UW473 e ONS-76. Observou-se um aumento da expressão proteica da ?-catenina, da Aurora A e B na UW473, o que ocorreu somente com a ?-catenina na linhagem ONS-76. Após o tratamento com o AMG900 ocorreu uma diminuição da expressão proteica de ?-catenina, da Aurora A e B em ambas as linhagens. A transfecção não alterou o percentil celular em G2/M na UW402 e UW473. Já na ONS-76 houve um aumento significativo em G2/M, e o AMG900 potencializou esse bloqueio apenas nessa linhagem. Os resultados sugerem que pode haver alguma relação entre a inibição das proteínas Aurora-quinases e a expressão de proteínas da via Wnt. / Medulloblastoma (MB) is the most common malignant brain tumor in childhood. Tumor formation/progression has been associated to molecular alterations that include dysregulation of signaling pathway Wingless (Wnt), responsible for embryonic development. In addition, cell cycle proteins Aurora-kinase (A, B and C) have been widely studied since Aurora A and B were found overexpressed in many cancers such as MB. Recent studies show that there is an association between Wnt pathway and Aurora kinase proteins. However, few studies have been conducted to confirm this association. Moreover, there are no studies reporting the effects of AMG900 in MB, by focusing on the regulation of the Wnt pathway. The aim of this study is to evaluate Wnt pathway modulation by Aurora kinases inhibitor AMG900 in pediatric medulloblastoma cell lines. Conventional PCR, sequencing, qRT-PCR, transient transfection, clonogenic assay, Western Blot and cell cycle assays were performed. UW402, UW473 and ONS-76 cell lines did not present mutations in exon 3 of CTNNB1 gene and exon 15 of APC gene. There was no significant expression of CTNNB1 and their target genes in these cell lines, confirming that they did not have Wnt pathway activated. Considering this, transient transfection was necessary. After this trial, there was an increase in expression of CTNNB1 gene and its target genes Cyclin D1 and C-Myc in the three cell lines, which was not observed in Aurora kinases. Furthermore, in the clonogenic assay, a reduction in the number of colonies in UW473 and ONS-76 cell lines was observed. It was also observed an increase in ?-catenin protein, Aurora A and B in UW473 cell line, but not in ONS-76 cell line. However, after treatment there was a decrease in protein expression of ?-catenin, Aurora A and B in both cells. Transfection did not change the cellular percentile in G2 / M in UW402 and UW473. In ONS-76 there was a significant increase in G2 / M, and the treatment with AMG900 potentiated this block only in this cell line. Results suggest that there may be some relation between the inhibition of Aurora kinase protein and protein expression in Wnt pathway.
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Mecanismos Envolvidos com a Inibição de Aurora-Quinases em Carcinoma de Adrenal / Mechanisms Involved in the Inhibition of Aurora Kinases in Adrenal CarcinomaKleiton Silva Borges 09 May 2014 (has links)
Introdução: Tumores adrenocorticais (TAC) são raros, correspondendo somente a 0,2% de todas as neoplasias pediátricas, sendo que a maioria dos casos são diagnosticados no Brasil e estão associados com a mutação TP53 p.R337H. A cirurgia é o único tratamento efetivo conhecido para os TAC, sendo os tumores em estadios avançados frequentemente fatais. A família das Aurora-quinases é formada por três membros (Aurora-A, -B e -C) os quais atuam em diversas fases do ciclo celular, como alinhamento dos cromossomos, formação do fuso mitótico e citocinese. Diferentes trabalhos mostraram a expressão alterada de membros desta família em vários tipos de tumores e a inibição da atividade destas proteínas tem sido considerada uma potencial abordagem para o tratamento do câncer. Objetivo: A partir da análise da expressão dos genes Aurora-A e Aurora-B em amostras de TAC pediátrico, foram investigados os efeitos do AMG 900, um pan-inibidor de aurora quinases, na proliferação, apoptose, síntese hormonal e perfil transcricional da linhagem H295A. Além disso, foram avaliados os efeitos do AMG 900 combinado com diferentes quimioterápicos. Metodologia: Os níveis de expressão dos genes Aurora-A e Aurora-B foram analisados em 60 crianças com TAC através das técnicas de RT-qPCR e imuno-histoquímica. A proliferação celular foi avaliada por coloração com Giemsa e a apoptose foi realizada por citometria de fluxo. A análise de combinação de drogas foi feita com base no método de Chou-Talalay e o ensaio de microarray foi realizado utilizando a plataforma da Agilent. Resultados: A expressão dos genes Aurora-A e Aurora-B foi associada com estadios avançados da doença e a expressão do Aurora-A foi associada com a presença da mutação TP53 p.R337H. O tratamento com o AMG900 causou a inibição da proliferação, aumento da apoptose e sensibilizou as células para os inibidores de topoisomerase II (doxorrubicina e etoposídeo). Adicionalmente, o AMG 900 levou à redução da síntese de hormônios bem como modulou a expressão de genes envolvidos com esta atividade. A inibição das aurora-quinases alterou a expressão de genes associados com a regulação da fase G1 do ciclo celular e afetou a expressão de genes da via de sinalização Notch. Conclusão: A inibição das aurora-quinases pelo AMG 900 pode ser uma alternativa para o tratamento dos tumores adrenocorticais. / Introduction: Pediatric adrenocortical tumors (ACT) are rare malignancies representing only 0.2 % of all pediatric cancers. Most cases are diagnosed in Brazil and are associated with TP53 p.R337H mutation. Surgery is the only effective treatment known for the ACT however this approach has a small impact on survival in advanced disease. The Aurora kinase family is comprised of three members (Aurora-A, -B and -C) which act at different phases of the cell cycle, such as chromosomes alignment, mitotic spindle formation and cytokinesis. Several studies have demonstrated altered expression of members of this family in various types of tumors and the functional inhibition of the aurora kinases have been considered as a potential approach to cancer treatment. Aim: On the basis of analysis of Aurora-A and Aurora-B gene expression in the samples from pediatric ACT, we investigated the effects of AMG 900, a pan-aurora kinase inhibitor, on proliferation, apoptosis rate, hormone synthesis and transcriptional profile of H295A cell line. Furthermore, we evaluated the effects of AMG 900 combined with different chemotherapeutic agents. Methods: The mRNA expression levels of Aurora-A and Aurora-B genes were analyzed in 60 children with ACT by RT-qPCR and immunohistochemistry. Cell proliferation was assessed by Giemsa staining and apoptosis was performed by flow cytometry. Drug combination analysis was made on the basis of Chou- Talalay method. Microarray experiments were carried out using the Agilent human microarray. Results: Aurora-A and Aurora-B overexpression was associated with advanced disease. Patients carrying the TP53 p.R337H mutation presented significantly higher expression values of Aurora-A. Treatment with AMG900 caused inhibition of proliferation, increased apoptosis and sensitized the cells to topoisomerase II inhibitors (doxorubicin and etoposide). Additionally, the AMG 900 led to decreased synthesis of hormones and modulated the expression of genes involved in this activity. Finally, Aurora kinases inhibition altered the expression of genes associated with G1 cell cycle phase regulation and affected the Notch signaling pathway target genes. Conclusion: These data suggest that Aurora kinase inhibition by AMG900 may be a new therapeutic approach to adrenocortical carcinoma treatment.
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Co-targeting aurora kinase with PD-L1 and PI3K abrogates immune checkpoint mediated proliferation in peripheral T-cell lymphoma: a novel therapeutic strategyIslam, Shariful, Vick, Eric, Huber, Bryan, Morales, Carla, Spier, Catherine, Cooke, Laurence, Weterings, Eric, Mahadevan, Daruka 01 November 2017 (has links)
Peripheral T-cell non-Hodgkin lymphoma (PTCL) are heterogeneous, rare, and aggressive diseases mostly incurable with current cell cycle therapies. Aurora kinases (AKs) are key regulators of mitosis that drive PTCL proliferation. Alisertib (AK inhibitor) has a response rate similar to 30% in relapsed and refractory PTCL (SWOG1108). Since PTCL are derived from CD4(+)/CD8(+) cells, we hypothesized that Program Death Ligand-1 (PDL1) expression is essential for uncontrolled proliferation. Combination of alisertib with PI3K alpha (MLN1117) or pan-PI3K inhibition (PF-04691502) or vincristine (VCR) was highly synergistic in PTCL cells. Expression of PD-L1 relative to PD-1 is high in PTCL biopsies (similar to 9-fold higher) and cell lines. Combination of alisertib with pan-PI3K inhibition or VCR significantly reduced PD-L1, NF-kappa B expression and inhibited phosphorylation of AKT, ERK1/2 and AK with enhanced apoptosis. In a SCID PTCL xenograft mouse model, alisertib displayed high synergism with MLN1117. In a syngeneic PTCL mouse xenograft model alisertib demonstrated tumor growth inhibition (TGI) similar to 30%, whilst anti-PD-L1 therapy alone was ineffective. Alisertib + anti-PD-L1 resulted in TGI > 90% indicative of a synthetic lethal interaction. PF-04691502 + alisertib + anti-PD-L1 + VCR resulted in TGI 100%. Overall, mice tolerated the treatments well. Co-targeting AK, PI3K and PD-L1 is a rational and novel therapeutic strategy for PTCL.
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Caractérisation moléculaire et fonctionnelle des protéines GIPs (Gamma-tubulin complex protein 3-Interacting Proteins) d'Arabidopsis thaliana / Molecular and functional characterization of proteins GIPs (Gamma-tubulin complex protein 3-interacting proteins) in Arabidopsis thalianaMasoud, Kinda 25 January 2013 (has links)
Les microtubules constituent l’un des réseaux du cytosquelette des cellules eucaryotes. Ils jouent un rôle central dans de multiples fonctions comme la division cellulaire, les trafics intracellulaires et la morphogenèse cellulaire. Chez les plantes supérieures, les microtubules (MTs) forment différents réseaux qui s'assemblent au cours du cycle cellulaire. Cette spécificité nécessite un recrutement régulé des complexes de nucléation des MTs à l’enveloppe nucléaire, au cortex et au niveau de MTs préexistants, qui sont des sites de nucléation caractérisés. L'équipe d’A.C. Schmit (IBMP, CNRS, Strasbourg), dans laquelle j'ai effectué mon travail de thèse, se focalise sur la caractérisation des complexes de nucléation des MTs (γ-TuRCs) et la régulation de l'assemblage du fuseau mitotique chez les plantes. Deux nouvelles protéines associées au γ-TuRC ont été mises en évidence par une interaction directe avec l'un de ses composants AtGCP3. Ces protéines, AtGIP1 et AtGIP2 (GCP3 Interacting Protein 1 et 2), sont très conservées au cours de l'évolution, mais leur fonction reste totalement inconnue. Mon travail a été consacré à la caractérisation de cette nouvelle classe de protéines dans le but de comprendre leur rôle. Nos résultats suggèrent que l'association des protéines GIPs aux γ-TuRCs participe à la régulation de leur activité et à la formation d'un fuseau mitotique robuste. Le profil de localisation des protéines GIPs au cours du cycle cellulaire et les phénotypes observés chez les mutants "perte de fonction" gip1gip2 indiquent que ces protéines interviennent dans le recrutement des γ-TuRCs, la nucléation des MTs, l’assemblage du fuseau mitotique, le déroulement du cycle cellulaire et l'organisation des méristèmes. L’étude des mécanismes de régulation de cette famille de protéines a été initiée. Nos résultats ont permis d’identifier GIP1comme un substrat de la kinase Aurora1 in vitro. Les résultats d’expérience de complémentation avec des phosphomutants GIP1 indiquent que la/les fonction(s) des GIPs pourrai(en)t être dépendante(s) de la phosphorylation par la kinase Aurora1, qui est un régulateur avéré du cycle cellulaire. L’ensemble de mes travaux a ainsi contribué à la caractérisation de nouveaux acteurs du cytosquelette microtubulaire. Une meilleure connaissance de leur réseau d'interaction (interactome) ainsi que l’étude de leur homologue humain pourraient ouvrir de nouvelles perspectives de recherche dans le contrôle de la division cellulaire et la lutte contre le cancer. / Microtubules (MTs) constitute one of the cytoskeletal networks in eukaryotic cells. They are involved in various processes such as cell division, intracellular transport and cell morphogenesis. In higher plants, MTs can be organized into dynamic structures, which undergo continual assembly and disassembly during the cell cycle. This specificity requires the recruitment of the nucleation complexes of the MTs to the nuclear envelope, to the cortex and to pre-existing MTs. The work of A. C. Schmit’s team (IBMP, CNRS, Strasbourg), in which I did my thesis, focuses on the characterization of MT nucleation complexes (γ-TuRCs) and the regulation of mitotic spindle assembly in plants. We have identified small proteins interacting with Gamma-tubulin Complex Protein 3 (GCP) and named GIP1 and GIP2 (GCP3-Interacting Proteins). The aim of these studies was to characterize this new class of proteins in order to understand their role. It shows that GIPs are conserved among eukaryotes and suggests that their association with the γ-TuRC participates in the regulation of their activity and the formation of a robust mitotic spindle. The localization of GIPs during the cell cycle and the phenotypes observed in T-DNA insertional gip1gip2 double mutants indicatethat GIPs are required for the recruitment of γ-TuRCs, MT nucleation, spindle assembly, cell cycle regulation and stem cell maintenance. Likewise, in vitro assays showed that GIP1 is a novel substrate for Aurora kinase1, which is a well known cell cycle regulator. The results of complementation experiments with GIP1 phosphomutants indicate that the phosphorylation of GIPs may be required for their function(s). Altogether, our results have contributed to the characterization of a new class of proteins involved in MT nucleation/organization and functions. The study of the interaction network (interactome) of GIPs and oftheir homologues could open new ways of research in the control of cell division and in the fight against cancer.
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