TRANSMISSION OF MUTANT p53 AGGREGATES LEAD TO MUTANT P53-LIKE PHENOTYPE.

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  • Presentation type: Iniciação Científica
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  • Keywords: p53; mutant; amyloid oligomer;
  • 1 Universidade Federal do Rio de Janeiro

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Abstract
Introduction and objective: Cancer is the main cause of death worldwide. There cellular mechanisms that are used to prevent it, one of them is the activation of the tumor suppressor p53 protein, which is known as one of the major cell cycle regulating agents. It has great importance in cell quality control, regulating death, senescence and replication. However, many mutations in the TP53 gene can promote poor protein folding, less stability and can lead to the formation of amyloid aggregates. Mutations often lead to two different situations: loss of wild-type protein function or gain of oncogenic function promoted by the mutant form. In gain-of-function cases, this protein acquires characteristics that lead to tumor growth. Loss of function leads to poor regulation of important processes such as DNA repair, cell cycle control and apoptosis. In this work, we seek to study the influence of mutant p53 cells on wild-type p53 (WTp53) cells, mimetizing the tumor microenvironment. Material and methods: The cell lines used in this work were MDA-MB-231 and OVCAR-3 (which express R280K and R248Q p53 mutant, respectively), and MCF-7 and A2780 (Wtp53 cell lines). We used western blotting for protein levels assessment, confocal fluorescence microscopy to detect protein accumulation and amyloid oligomers, optical microscopy to evaluate cell morphology, MTT assay to analyze the cell viability, clonogenic assay to evaluate the anchoring and colony formation and the wound healing assay to visualize the migration pattern of the cells. Results and conclusion: We observed that when we treated MCF-7 cells with the total extract, or conditioned medium (CM) from MDA-MB-231 cells, p53 accumulates more and colocalizes in confocal fluorescence microscopy with amyloid oligomers stained with A11 antibody, suggesting an incorporation of aggregates from the CM or donor cells extract, indicating a prion-like behavior. They also seem to display an epithelial-to-mesenchymal transition when evaluated by light microscopy, acquiring morphological characteristics similar to mutant donor cells. The treatments did not interfere in the cell viability of MCF-7 cells evaluated by MTT assay or their proliferation capacity after 48 h of treatment by Trypan blue counting. We also noticed that the treated cells increased cell migration in the wound healing assay. Furthermore, there is a decrease in the anchorage and colony formation capacity of the treated cells. When we treated cells with recombinant p53 aggregates, we observed by western blotting assay, increased levels of p53. The results obtained indicate a transfer or incorporation of p53 aggregates by WTp53 cell lines. Now, we are trying to elucidate the molecular mechanisms of cell-to-cell transfer of these aggregates in order to find a more specific pharmacological target for antitumor therapy.
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Author

Nathalia Oliveira da Silva

Boa tarde, Igor! Obrigada! Vou responder suas questões a seguir: 1) A partir desse resultado nós tivemos a mesma dúvida. Já foi reportado na literatura a transição epitélio-mesenquimal na presença de p53 mutante, mas nosso objetivo agora é fazer esse experimento novamente com uma linhagem de MDA-MB-231 silenciada para p53. Dessa forma poderemos ver se esse efeito de fato é dependente de p53 mutante ou não. 2) Não, nunca fizemos experimentos com reguladores. Mas, é uma excelente sugestão! podemos avaliar isso futuramente. Obrigada 3) Na literatura foi descrito que as células recebem esses agregados por macropinocitose. Nós também queremos isolar e avaliar o conteúdo dos exossomas presentes nas culturas pois acreditamos que eles são liberados no microambiente tumoral e podem conter agregados de p53 mutante. Se houverem mais dúvidas, me coloco à disposição para esclarecê-las Att. Nathalia Oliveira

Igor Petrone

Obrigado pelos esclarecimentos e, mais uma vez, parabéns pelo excelente trabalho!

Igor Petrone

Obrigado pelos esclarecimentos e, mais uma vez, parabéns pelo excelente trabalho!