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Luciana Maria Silvahttp://lattes.cnpq.br/2580791289874240Letícia da Conceição BragaGeovanni Dantas CassaliGeovanni Dantas CassaliEnrrico BloiseHelen Lima Del Puertohttp://lattes.cnpq.br/7161584290291649Milene Pereira Moreira2019-10-01T12:53:17Z2019-10-01T12:53:17Z2018-02-26http://hdl.handle.net/1843/30165O câncer de mama triplo-negativo (TNBC) caracteriza-se por uma alta heterogeneidade molecular e celular, o que influencia na resposta terapêutica e dificulta a descoberta de alvos efetivos. Essa heterogeneidade é atribuída à presença de células-tronco do câncer (CSCs) de mama, o que determina a resistência à quimioterapia, reduzindo a eficácia do tratamento e, subsequente recorrência da doença e metástase. Neste contexto, este trabalho teve como objetivo avaliar a heterogeneidade celular morfológica e fenotípica e a expressão de genes da via de sinalização do EGFR em subpopulações celulares obtidas de linhagens de TNBC, bem como suas implicações na resistência à quimioterapia. Para isso as linhagens de câncer de mama BT-549 e Hs 578T e a linhagem de tumor benigno de mama HMT-3522 S1 foram cultivadas em monocamada e em cultura de enriquecimento de CSCs (ensaio de tumoresferasTS). Em seguida, BT-549 and Hs 578T foram caracterizadas por microscopia de fluorescência e eletrônica e citometria de fluxo. Além disso, as células foram tratadas com paclitaxel (PTX) e doxorrubicina (DOX). Para o estudo de expressão de genes associados à via EGFR foi utilizado a BT-549 TS antes e após o tratamento com DOX. Os resultados mostraram que a BT-549 TS após enriquecimento de CSCs, identificada pelo fenótipo CD44+/CD24- e ALDH+ apresentou subexpressão da maioria dos genes avaliados (64 genes) e apenas 3 genes foram superexpressos. Entre os genes superexpressos (MAPK3, PRKCZ e STAT3), STAT3 apresentou o maior nível de expressão. A análise da BT-549 e Hs 578T demonstrou grande heterogeneidade celular entre as linhagens, bem como entre as subpopulações celulares que as compõem, tanto em termos morfológicos quanto fenotípicos e de resposta a PTX e DOX. Para BT-549, sugere-se que a plasticidade das células tumorais híbridas CD44+/CD24+/CD146+, caracterizadas por apresentar um fenótipo mesenquimal e epitelial, e das células tumorais epiteliais CD44-/CD24+/ CD146+ originou a CSCs EMT-like CD44+/CD24-/CD146-, através da conversão fenotípica induzida por transição epitélio-mesenquimal (EMT). Além disso, estas células podem gerar CSCs identificadas como ALDH+ epitelial-like. As CSCs podem se autorrenovar e diferenciar em células tumorais (CD44-/CD24-/CD146-). Esta plasticidade fenotípica não foi observada para a Hs 578T e HMT-3522S1. Os dados de citotoxicidade mostraram que a BT-549 foi mais resistente ao PTX, o que parece estar associado ao fenótipo CD24+. Em contraste, a BT-549 TS foi extremamente resistente a DOX, devido ao enriquecimento de CSCs (CD44+/CD24-/CD146- e ALDH+). Esses resultados mostram a alta heterogeneidade e plasticidade associada à EMT da BT-549, sendo esta capaz de alterar completamente o fenótipo das subpopulações celulares sob cultura de enriquecimento de CSCs. Esse achado parece desempenhar um papel importante na resistência de TNBC ao tratamento com DOX. Além disso, STAT3 destacou-se como uma assinatura molecular de TNBC relacionada à resistência a DOX e como possível alvo terapêutico.Triple negative breast cancer (TNBC) is characterized by high molecular and cellular heterogeneity, which influences the therapeutic response and it makes difficult the discovery of effective targets. This heterogeneity is attributed to the presence of breast cancer stem cells (BCSCs), which determines resistance to chemotherapy and subsequently disease recurrence and metastasis. In this context, this work aimed to evaluate the morphological and phenotypic cellular heterogeneity and the EGFR signaling pathway gene expression in cell subpopulations obtained from TNBC cell lines, as well as determined their implications in chemotherapy resistance. The BT-549 and Hs 578T TNBC cell lines and HMT-3522 S, a nonmalignant breast cell line, were cultured in monolayer and in BCSCs enrichment culture (tumorspheres-TS assay). Furthermore, the BT-549 and Hs578T were characterized by fluorescence and electron microscopy and flow cytometry. In addition, the cell lines cultured in two models were treated with paclitaxel (PTX) and doxorubicin (DOX). BT-549 TS was used to evaluate EGFR-associated gene expression before and after DOX treatment. The results showed that BT-549 TS after BCSCs enrichment, identified by the CD44+/CD24- and ALDH+ phenotype, showed subexpression a majority of the evaluated genes (64 genes) and only 3 genes were overexpressed. Among the overexpressed genes (MAPK3, PRKCZ and STAT3), STAT3 had the highest expression level. The BT-549 and Hs 578T analysis demonstrated large morphological and phenotypic heterogeneity between these cell lines, as well as between the cell subpopulations that compose them, as well as in PTX and DOX response. For BT-549, it is suggested that the plasticity of CD44+/CD24+/CD146+ hybrid tumor cells, characterized by a mesenchymal and epithelial phenotype, and CD44/CD24+/CD146+ epithelial tumor cells originated CD44+/CD24-/CD146- BCSCs EMT-like, through the phenotypic conversion induced by epithelial-mesenchymal transition (EMT). In addition, these cells could originate BCSCs identified ALDH+ epithelial-like. BCSCs can selfrenewal and differentiate into CD44-/CD24-/CD146- tumor cells. Same phenotypic plasticity was not observed for Hs 578T and HMT-3522 S1. Cytotoxicity data showed that BT-549 was more resistant to PTX, which appears to be associated with the CD24+ phenotype. In contrast, BT-549 TS was extremely resistant to DOX probably due to the enrichment of BCSCs (CD44+/CD24-/CD146- and ALDH+ cells). These results showed the high BT-549 EMTassociated heterogeneity and plasticity, being able to completely altered the phenotype of the cellular subpopulations under CSC enrichment culture. This finding appears to play an important role in TNBC resistance to DOX treatment. In addition, STAT3 was highlighted as DOX-resistance related TNBC molecular signature and as a possible therapeutic target.porUniversidade Federal de Minas GeraisPrograma de Pós-Graduação em PatologiaUFMGBrasilMEDICINA - FACULDADE DE MEDICINANeoplasias da MamaCélulas-Tronco NeoplásicasHeterogeneidade GenéticaTratamento FarmacológicoResistencia a Medicamentos AntineoplásicosCâncer de mamaCélula-tronco do câncerHeterogeneidade celularQuimioterapiaResistênciaAnálise da heterogeneidade celular estrutural e ultraestrutural, imunofenotipagem e via de sinalização EGRF em subpopulações de células-tronco de linhagens de câncer de mama triplo- negativo e suas implicações no mecanismo de resistência quimioterápicainfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFMGinstname:Universidade Federal de Minas Gerais (UFMG)instacron:UFMGORIGINALTESE 2018_Milene Pereira Moreira.pdfTESE 2018_Milene Pereira Moreira.pdfAbertoapplication/pdf9788600https://repositorio.ufmg.br/bitstream/1843/30165/2/TESE%202018_Milene%20Pereira%20Moreira.pdf01c6b5522840e57ef7e8f87b1d98e017MD52LICENSElicense.txtlicense.txttext/plain; charset=utf-82119https://repositorio.ufmg.br/bitstream/1843/30165/3/license.txt34badce4be7e31e3adb4575ae96af679MD53TEXTTESE 2018_Milene Pereira Moreira.pdf.txtTESE 2018_Milene Pereira Moreira.pdf.txtExtracted texttext/plain207942https://repositorio.ufmg.br/bitstream/1843/30165/4/TESE%202018_Milene%20Pereira%20Moreira.pdf.txt928287fb791a8729defb5cead2adc16eMD541843/301652019-11-14 12:37:43.373oai:repositorio.ufmg.br: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Repositório InstitucionalPUBhttps://repositorio.ufmg.br/oaiopendoar:2019-11-14T15:37:43Repositório Institucional da UFMG - Universidade Federal de Minas Gerais (UFMG)false
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