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TALITA HELEN BOMBARDELLI GOMIG LAZAROTTO.Pdf UNIVERSIDADE FEDERAL DO PARANÁ TALITA HELEN BOMBARDELLI GOMIG LAZAROTTO ANÁLISE PROTEÔMICA COMPARATIVA NO CÂNCER DE MAMA: PREDIÇÃO DE VIAS E FUNÇÕES BIOLÓGICAS E DE INTERAÇÕES PROTEICAS BASEADA EM ESPECTROMETRIA DE MASSA E ANÁLISES DE BIOINFORMÁTICA CURITIBA 2019 TALITA HELEN BOMBARDELLI GOMIG LAZAROTTO ANÁLISE PROTEÔMICA COMPARATIVA NO CÂNCER DE MAMA: PREDIÇÃO DE VIAS E FUNÇÕES BIOLÓGICAS E DE INTERAÇÕES PROTEICAS BASEADA EM ESPECTROMETRIA DE MASSA E ANÁLISES DE BIOINFORMÁTICA Tese apresentada ao curso de Pós-Graduação em Genética, Setor de Ciências Biológicas, Universidade Federal do Paraná, como requisito parcial à obtenção do título de Doutor em Genética. Orientador: Prof. Dr. Iglenir João Cavalli Coorientador(a): Prof(a). Dr(a). Enilze Maria de Souza Fonseca Ribeiro CURITIBA 2019 AGRADECIMENTOS Primeiramente, à Deus, por cada oportunidade e por todos em minha vida. Aos meus orientadores, pelos ensinamentos transmitidos e construídos em nossos dez anos de convivência, e pela oportunidade de crescimento pessoal e profissional que têm proporcionado. Agradeço pela confiança em mim e em meu trabalho desde os tempos da Iniciação Científica e, em especial, pela dedicação para com minha formação e pela grande amizade que permanece. A todos os professores, pesquisadores, técnicos e demais profissionais que contribuíram de variadas formas para o meu aprendizado e para a execução deste e de outros estudo dos quais participei. À todas as instituições envolvidas e seus integrantes e, em particular, aos pacientes que aceitaram participar dessa e de outras pesquisas científicas, à equipe médica e aos funcionários dos hospitais. E ao apoio financeiro das agências de fomento, CAPES, CNPq e Fundação Araucária. Aos membros da banca, pelo aceite em participar da defesa de tese e por contribuírem com seus conhecimentos e reflexões. Aos integrantes do Laboratório de Citogenética Humana e Oncogenética, antigos e atuais, pela amizade, apoio e convivência. Em especial, às pessoas que dedicaram seu tempo e seus esforços para me ajudar em experimentos e interpretações de resultados. De forma singular, agradeço com muito carinho às amizades concretizadas ao longo desses anos... Sem vocês, a rotina de trabalho e a vida não seriam as mesmas. Obrigada por partilharem de tantos momentos, histórias, loucuras e desafios. À minha família de amigos, pelos momentos de descontração, encontros, conversas e risadas, especialmente por se fazerem presentes mesmo em minhas ausências. Agradeço a compreensão e o apoio de sempre. Em especial e com muito amor e carinho, agradeço aos meus familiares e à minha família, pela parceria e por todo o suporte, incentivo e confiança em meus passos ao longo desses anos de dedicação à pesquisa. Obrigada pelo amor e carinho, e por cada exemplo, oportunidade e ensinamento propiciados. A gratidão por ter vocês em minha vida é imensurável. A tudo e a todos que contribuíram, direta ou indiretamente, afetiva ou profissionalmente, para a minha formação e conclusão deste trabalho, meus sinceros agradecimentos! “First, think. Second, believe. Third, dream. And finally, dare.” Walt Disney RESUMO O câncer de mama é o mais comum em mulheres no mundo e o segundo mais incidente em mulheres no Brasil, após o câncer de pele não melanoma. Em homens, é uma malignidade pouco frequente representando cerca de 1,8% dos casos de câncer de mama em geral. Tecnologias de alta resolução aliadas à bioinformática têm sido utilizadas como estratégias de estudo para auxiliar no diagnóstico, na seleção da terapia e no prognóstico do câncer. Nesse contexto, as análises de proteoma e interatoma consistem em importantes abordagens para evidenciar os principais processos biológicos associados à tumorigênese, uma vez que níveis de expressão proteica alterados podem influenciar vias celulares críticas e redes de interações, e comprometer a homeostase do ambiente tecidual, contribuindo para o desenvolvimento do câncer. Neste estudo, a espectrometria de massa baseada na quantificação livre de marcação, seguida da anotação/enriquecimento funcional e da predição de redes de interação proteína- proteína, foi realizada para obter um panorama do proteoma diferencial e seu contexto biológico funcional no câncer de mama feminino e masculino. Os níveis de expressão proteica dos tumores primários de mama foram avaliados simultaneamente aos linfonodos axilares metastáticos e tecidos mamários não tumorais para identificar as proteínas diferencialmente expressas entre esses tecidos. Análises in sílico dos dados proteômicos foram utilizadas para identificar as vias de sinalização e metabólicas, funções e processos biológicos mais relevantes, bem como redes de interação que envolvem proteínas com alteração de expressão. Os dados referentes à análise do câncer de mama feminino demonstraram um alto nível de similaridade no perfil proteômico de ambos os tecidos mamários não tumorais (contralateral e adjacente) e entre os malignos (tumor primário de mama e linfonodo axilar metastático). O contexto biológico geral dessa análise incluiu as vias de LXR/RXR, óxido nítrico, eNOS, eIF2 e sirtuínas; metabolismo de ácidos graxos; estresse oxidativo; funções relacionadas ao tumor; e o processo metastático; além de funções biológicas agrupadas nos hallmarks clássicos do câncer e em fenótipos de estresse, processos metabólicos e vias de sinalização. O proteoma comparativo do câncer de mama masculino, por sua vez, evidenciou várias proteínas diferencialmente expressas entre os tecidos analisados. Em geral, essa análise identificou as vias de sinalização de granzima B, sirtuínas, eIF2, citoesqueleto de actina, eNOS, cálcio e resposta de fase aguda relacionadas às proteínas com alteração de expressão. A análise proteômica comparativa adicional, realizada entre os tecidos de tumor mamário masculino e o de uma paciente do sexo feminino com o mesmo subtipo da doença e parâmetros clinicopatológicos similares, indicou um relevante conjunto de proteínas diferencialmente expressas, envolvido principalmente em processos relacionados ao câncer e na transdução de sinal. Ao todo, este estudo revela perfis proteômicos diferenciais em processos associados e interconectados de sinalização no câncer de mama feminino e masculino e fornece fontes de dados relevantes para a pesquisa do câncer, as quais podem contribuir para procedimentos clínicos no câncer de mama. Para isso, abordagens complementares e estudos de validação são recomendados para confirmar o potencial de proteínas individuais e/ou de vias como marcadores biológicos na tumorigênese mamária. Palavras-chave: Câncer de mama. Proteômica. LFQ-MS. Bioinformática. ABSTRACT Breast cancer is the most common cancer in women worldwide and the second most incident in women in Brazil, after non-melanoma skin cancer. In men, it is a rare malignancy accounting for about 1.8% of overall breast cancer cases. High- throughput technologies allied to bioinformatics have been used as study strategies to assist in the diagnosis, therapy selection, and prognosis of cancer. In this context, proteome and interactome analyses consist in important approaches to highlight the main biological processes associated with tumorigenesis, since the protein levels altered expression can influence critical cellular pathways and interactive networks, and compromise the tissue environment homeostasis contributing to cancer development. In this study, the mass spectrometry-based label-free quantification followed by functional annotation/enrichment and protein-protein interaction networks prediction were performed to obtain a landscape of the differential proteome and its functional biological context in the female and male breast cancer. Protein expression levels of the primary breast tumors were evaluated simultaneously with the corresponding metastatic axillary lymph nodes and the non-tumor breast tissues to identify the differentially expressed proteins among these tissues. In silico analyses of the proteomic data were used to identify the most relevant signaling and metabolic pathways, biological functions and processes as well as interaction networks related to the deregulated proteins. Data from female breast cancer analysis demonstrated a high level of similarity in the proteome profile of both non- tumor breast tissues (contralateral and adjacent) and between the malignant ones (primary breast tumor and axillary metastatic lymph nodes). The general biological context of this analysis included the pathways of LXR/RXR, nitric oxide, eNOS, eIF2 and sirtuins; fatty acid metabolism; oxidative stress; tumor-related functions; metastatic process; and to biological roles grouped into the classic hallmarks of cancer and in stress phenotypes, metabolic processes and signaling pathways. The comparative proteome of male breast cancer, on the other hand, highlights various differentially expressed proteins among the tissues analyzed. In general, this analysis identified the signaling pathways of granzyme B, sirtuins, eIF2, actin cytoskeleton, eNOS, calcium and acute phase response related to the deregulated proteins. The additional proteomic comparative analysis, performed between the tissues of the male breast tumor and the ones of a female patient matched for the breast cancer subtype and similar clinico-pathological parameters, indicated an interesting set of differentially expressed proteins, which were mainly involved in cancer-related biological processes and signal transduction. Altogether, this study reveals differential proteomic profiles in cancer associated and interconnected
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