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Breast cancer is the most common and fatal type of cancer in women worldwide due to the metastatic process and resistance to treatment. Despite advances in molecular knowledge, little is known regarding resistance to chemotherapy. One highlighted aspect is the DNA damage response (DDR) pathway that is activated upon genotoxic damage, controlling the cell cycle arrest or DNA repair activation. Recently, studies have showed that cancer stem cells (CSCs) could promote chemoresistance through DDR pathway. Furthermore, it is known that the epithelial-mesenchymal transition (EMT) can generate cells with CSCs characteristics and therefore regulate the chemoresistance process. The exosomes are microvesicles filled with RNAs, proteins and microRNAs (miRNAs) that can be released by many cell types, including tumor cells and CSCs. The exosomes content may be cell-to-cell transferable and it could control a wide range of pathways during tumor development and metastasis. A big challenge for modern medicine is to determine the reasons why patients do not respond to chemotherapy treatments and also guide the most appropriate therapy for each one. Considering that the CSCs are able to stimulate the formation of a more aggressive tumor phenotype with migration and metastasis ability, resistance to treatment and disease recurrence, as well as few studies capable to determine clearly the interaction of breast CSCs with its microenvironment, the present review summarize the possibility that exosomes-mediate miRNAs transfer and regulate chemoresistance in breast tumor cells and CSCs, to clarify the complexity of breast cancer progression and therapy.

Orientador: Marcelo Lima Ribeiro Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Diariamente milhares de eventos moleculares danificam o DNA de nossas células. Para contrapor tais modificações existem eficientes vias de reparo ao DNA. A maquinaria de reparo do DNA desempenha um papel fundamental na manutenção da estabilidade genômica evitando que mutações ocorram. As enzimas de reparo ao DNA monitoram continuamente o DNA para corrigir qualquer dano que possa ser causado por agentes mutagênicos exógenos e endógenos. Além de mutações, que podem ser herdadas ou adquiridas somaticamente, alterações epigenéticas em genes de reparo do DNA têm sido associadas à carcinogênese. O câncer gástrico (GC) representa a segunda maior causa de mortalidade por câncer em todo o mundo. A doença se desenvolve a partir do acúmulo de várias alterações genéticas e epigenéticas. Entre os fatores de risco a infecção por "Helicobacter pylori" ("H. pylori") é considerado o principal fator associado ao desenvolvimento da doença. Sabe-se que esta bactéria induz a perda de características importantes na diferenciação epitelial, favorecendo a transição para um fenótipo mesenquimal, induz a formação de células tronco tumorais (CSCs) gástricas e, por conseguinte, a formação de tumores. As CSCs são resistentes ao tratamento de GC e, subsequentemente, responsável pela recorrência de tumores e metástases. Portanto, a determinação dos mecanismos de regulação e manutenção das CSCs são essenciais para compreender a fisiopatologia do GC. Assim, o presente estudo analisou o papel da infecção por "H. pylori" na regulação epigenética dos sistemas de reparo DSB e BER em células epiteliais e CSCs gástricas. Neste trabalho nós identificamos que a infecção por "H. pylori" estimula as principais características associadas à carcinogênese, tais como a proliferação celular, a translocação nuclear de 'beta'-catenina, indução de danos ao DNA e consequente indução de genes de reparo de forma dependente dos fatores de virulência bacteriano. Além disso, determinou-se que a indução do reparo ao DNA é mediada pela hiperacetilação das histonas H3 e H4 e hipometilação da região promotora. Determinou-se também que SOX9 é essencial para a manutenção das CSCs e estimula processos associados à carcinogênese gástrica, tais como proliferação celular, translocação nuclear de 'beta'-catenina, quimiorresistência, diminuição da apoptose e senescência. Além disso, encontramos uma interação em loop entre SOX9 e 'beta'-catenina. Interessantemente, demonstrou-se que SOX9 é induzido em resposta à "H. pylori" tanto em células epiteliais como em CSCs gástricas. Finalmente, observou-se que ATM está ativado em CSCs gástricas e infectadas por "H. pylori" e que SOX9 regula os genes de reparo ao DNA. A partir destes resultados, concluiu-se que a indução de reparo ao DNA ocorre em resposta à "H. pylori" para prevenir ou reduzir instabilidade genômica. Nossos resultados fornecem um novo mecanismo epigenético associado aos danos ao DNA causados pela infecção por "H. pylori" Abstract: Although thousands of DNA damaging events occur in each cell every day, efficient DNA repair pathways have evolved to counteract them. The DNA repair machinery plays a key role in maintaining genomic stability by avoiding the maintenance of mutations. The DNA repair enzymes continuously monitor the DNA to correct any damage that is caused by exogenous and endogenous mutagens. In addition to mutations, which can be either inherited or somatically acquired, epigenetic changes in DNA repair genes has been associated with carcinogenesis. Gastric cancer (GC) represents the second highest cause of cancer mortality worldwide. The disease develops from the accumulation of several genetic and epigenetic changes during the lifetime. Among the risk factors, "Helicobacter pylori" ("H. pylori") infection is considered the main driving factor to gastric cancer development. This bacteria induces the loss of key features of epithelial differentiation, the transition to a mesenchymal phenotype, endorses the transformation of gastric stem cells and therefore tumor formation. These cells with self-renewal properties (called cancer stem cells, CSCs) are also resistant to GC therapy and subsequently responsible for tumor recurrence and metastasis. Therefore, the determination of the mechanisms of CSCs regulation and maintenance are essential for understanding the pathobiology of GC. Thus, the current study analyzed the role of "H. pylori" infection on the epigenetic regulation of DSB and BER DNA repair systems in epithelial and CSCs. In this work we have identified that H. pylori infection stimulates key features of the development of cancer, such as cell proliferation, nuclear translocation of 'beta'-catenin, DNA damage and consequent induction of DNA repair genes response in a virulence factors dependent manner. In addition, it was determined that the induction of DNA repair is mediated by H3 and H4 hyperacetylation and hypomethylation of promoter region of certain genes. it was determined that SOX9 is essential for maintenance of CSCs and stimulate gastric key processes such as tumor cell proliferation, nuclear translocation 'beta'-catenin, chemoresistance, decreased apoptosis and senescence. In addition, we found a regulatory interaction loop between SOX9 and 'beta'-catenin. Interestingly, it was demonstrated that SOX9 is induced in response to "H. pylori" both in gastric epithelial cells as CSCs. Finally, it was observed that CSCs are regulated by ATM and there is an ATM increase in CSCs in response to H. pylori. From these results, we concluded that the DNA repair induction occurs in response to "H. pylori" to prevent or reduce chromosome aberrations. Our findings provide novel epigenetic mechanism associated to DNA damage caused by "H. pylori" infection Doutorado Genética Animal e Evolução Doutora em Genética e Biologia Molecular FAPESP 2011/21710-0

The DNA repair machinery plays a key role in maintaining genomic stability by preventing the emergence of mutations. Furthermore, the -93G>A polymorphism in the MLH1 gene has been associated with an increased risk of developing colorectal cancer. Therefore, the aim of this study was to examine the expression pattern and effect of this polymorphism in normal and tumour samples from patients with colorectal cancer. The MLH1 -93G>A (rs1800734) polymorphism was detected by PCR–RFLP in 49 cases of colorectal cancer. MLH1 expression was investigated using real-time quantitative PCR. The results indicate a significant decrease in MLH1 expression in tumour samples compared to their normal counterparts. The MLH1 gene was also significantly repressed in samples from patients who had some degree of tumour invasion into other organs. Similarly, those patients who were in a more advanced tumour stage (TNM III and IV) exhibited a significant reduction in MLH1 gene expression. Finally, the mutant genotype AA of MLH1 was associated with a significant decrease in the expression of this gene. This finding suggests that this polymorphism could increase the risk of developing colorectal cancer by a defective mismatch repair system, particularly through the loss of MLH1 expression in an allele-specific manner CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP Fechado