Orientador: Jörg Kobarg / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T05:36:59Z (GMT). No. of bitstreams: 1 MeloHanchuk_TalitaDiniz_D.pdf: 15551209 bytes, checksum: 4fffad9810f85e106f425f0bfbde833b (MD5) Previous issue date: 2015 / Resumo: Membros da família das Neks são cruciais para o início da mitose em eucariotos. Têm sido funcionalmente atribuídas a todas as 11 Neks humanas uma das três principais funções estabelecidas para esta família em mamíferos: (1) centríolos / divisão celular; (2) funções no cílio primário / ciliopatias; e (3) resposta à danos no DNA (DDR). No artigo de revisão (artigo I), relatamos uma análise detalhada atual sobre cada uma das 11 Neks. A hipótese é que as Neks possam conectar elementos reguladores que permitem o refinamento e a sincronização de eventos celulares. Dentre os membros desta família, Nek5 é a cinase mais negligenciada. Ensaios de duplo híbrido em leveduras (Y2H) foram realizados para identificar e caracterizar parceiros de interação Nek5; e proteínas mitocondriais foram observadas (artigo II). Ensaios de apoptose mostraram efeitos protetores na morte celular após tratamento com tapsigargina (2 ?M) de células HEK293T que superexpressam a hNek5, bem como a diminuição na formação de Espécies Reativas de Oxigênio após 4 horas de tratamento. A atividade da cadeia respiratória mitocondrial estava diminuída após superexpressão de hNek5, especialmente nas etapas de transferência de elétrons do TMPD para o citocromo c e no complexo II. O Y2H permitiu também a identificação da poliglutamilase de proteínas TTLL4 como um parceiro de Nek5 (artigo III). Células silenciadas para a Nek5, assim como células que expressam a versão "kinase dead" de Nek5, apresentaram por western blot e ensaio in vitro de atividade poliglutamilação um aumento na poliglutamilação de proteínas após transfecção com TTLL4. Em conclusão, nossos dados sugerem pela primeira vez a localização mitocondrial e a participação de Nek5 na morte celular e no processo poliglutamilação diminuindo a atividade de TTLL4 através de sua fosforilação inibitória / Abstract: Members of the Nek Family are crucial for the initiation of mitosis eukaryotes. All 11 human Neks have been functionally assigned to one of the three core functions established for this family in mammals: (1) centrioles/mitosis; (2) primary ciliary function/ciliopathies; and (3) DNA damage response (DDR). In the core section of the review (article I), we report the current detailed functional knowledge on each of the 11 Neks. We raise the hypothesis that Neks may be the connecting regulatory elements that allow the cell to fine tune and synchronize cellular events. Nek5 is the most neglected among members of the Nek kinases family. A yeast two-hybrid (Y2H) screen was performed to identify and characterize Nek5 interaction partners and mitochondrial proteins were retrieved (article 2). Apoptosis assay showed protective effects of hNek5 over-expression from Hek293-T¿s cell death after thapsigargin treatment (2 ?M) as well as an increase in ROS formation after 4 hours of treatment. Mitochondrial respiratory chain activity was found decreased upon hNek5 over-expression especially at the electrons transfer steps from TMPD to cytochrome c and at the complex II. The yeast two-hybrid allowed also the identification o TTLL4 as a Nek5 partner (article 3). Nek5 silenced cells as well as cells expressing a "kinase dead" version of Nek5, displayed an increase in polyglutamylation of proteins after TTLL4 transfection by western blot and in vitro polyglutamylation activity assay. In conclusion, our data suggest for the first time mitochondrial localization and functions for Nek5 and its participation in cell death and cell respiration regulation. This work also showed the function of Nek5 in the polyglutamylation process decreasing the role of TTLL4 through inhibitory phosphorylation by Nek5 / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

archives@tulane.edu / Triple negative breast cancers (TNBCs) are clinically and biologically aggressive, with higher recurrence and metastasis rates compared to other subtypes. Acquisition of a mesenchymal and migratory cell phenotype is consequential process that promotes metastasis. There are no clinically approved small molecule targeted therapies for TNBC; kinases are effective drug targets in cancer research. Although some kinases are known to regulate the mesenchymal phenotype, a large subset of the human kinome is understudied. There are many approaches to discovering novel kinase targets in cancer. Here, a phenotypic screen approach is described to identify understudied kinases using the Published Kinase Inhibitor Set (PKIS). Initial screens using TNBC cell lines (MDA-MB-231, BT549 and MDA-MB-157) identified 36 hits representative of twelve kinase inhibitor chemotypes based on reversal of the mesenchymal cell morphology. Our hits were further prioritized based on gene expression changes of the epithelial marker E-cadherin and migratory behavior. Active compounds were confirmed to reverse EMT on transcript and protein levels with qRT-PCR and Western blot. When pharmacologically similar compounds were more closely examined, different effects on cancer biology were observed (‘active’ versus ‘inactive’ compounds). Based on these observations, a kinase array was employed to compare both the active and inactive compounds to demonstrate how to identify candidate kinases responsible for the EMT reversal. Using this screening approach, small molecule inhibitors from the PKIS library (GSK346294A, GSK448459A, GSK237700A) were identified that were pharmacologically similar that reversed the mesenchymal phenotype in TNBC. These compounds have different biological effects in TNBC, despite having similar pharmacophores. Differential effects of the PKIS compounds on transwell migration, gene (qRT-PCR) and protein (Western blot) expressions, and mammosphere formation in TNBC cells was observed. In follow-up in vivo studies, our most active compound (GSK346294A) suppressed tumorigenesis and metastasis. RNA-sequencing confirmed downregulated pathways induced by GSK346294A treatment in TNBC cells included EMT, cytoskeletal rearrangement and cell cycle regulation. Because these compounds have different off-target activities, this approach can be used to identify candidate unique kinases responsible for the observed effects. NEK5 was one of these kinases candidates. NEK5 function remains understudied in cancer, and even more understudied in breast cancer. This study is the first, to our knowledge, to describe the function of NEK5 in breast cancer, specifically its roles in acquisition of a mesenchymal and migratory cell phenotype. Overexpression of NEK5 promotes a migratory and mesenchymal phenotype, and knockdown with a shRNA construct suppresses this migratory behavior. Data obtained using both qRT-PCR of the knockdown and overexpression cell lines, and follow-up RNA sequencing, revealed NEK5 regulates the PLAU/PAI-1/SRC axis. Furthermore, a role for NEK5 in resistance to SRC-targeting anticancer agents is demonstrated. The work described here demonstrates the utility of a novel approach to identify understudied kinases in cancer, and characterization of these kinases has potential impact in other metastatic diseases not limited to breast cancer. / 1 / Margarite Matossian