Dados do Trabalho

Título

Mutant p53 amyloid aggregates transmission in breast cancer

Introdução

Mutations in p53, a tumor suppressor protein, are present in more than 50% of cases of cancer. These mutations can promote the formation of amyloid oligomers and a prion-like effect, which the mutant protein converts the wildtype (WT) protein into an aggregated form and transmits the aggregates to other cells. The transmission of mutant p53 (mutp53) to other cells through extracellular vesicles (EVs) has been reported. However, it is not known whether p53 would be in the amyloid state, nor whether it has prion-like properties.

Objetivo

Thus, the aim of this study is to evaluate the transmission of mutant p53 amyloid aggregates among tumor cells.

Métodos

Breast cancer cell line, MCF-7 (WT p53), was treated with MDA-MB-231 (mutp53 R280K) conditioned medium (CM MDA) and the effects were evaluated using 2D and 3D cell culture models by western blotting (WB), dot-blot (DB), confocal fluorescence microscopy, immunoprecipitation, clonogenic assay and wound healing assay. Extracelular vesicles (EVs) isolated, by differential centrifugation, from MCF-7 (EVs M7) and MDA-MB-231 (EVs MDA) were characterized through dynamic light scattering (DLS), transmission electron microscopy (TEM), WB, and DB.

Resultados

MDA-MB-231 lysates showed proteinase k resistance, and induced the aggregation of WTp53, which may suggest a prion-like behavior. MCF-7 treated with CM MDA demonstrated higher levels of p53, amyloid oligomers and colocalization through confocal fluorescence microscopy, corroborating that p53 is in the amyloid state. Also, CM MDA promotes gains of function in the WTp53 cell line by increasing cell migration, reducing colony formation, disrupting 3D cell formation, and changing cell morphology. The characterization of the EVs from MCF-7 and MDA-MB-231 cells demonstrated size of 309.7 ± 51.8 and 392.7 ± 74.1 nm, respectively. The polydispersity index was 0.47 ± 0.07 (EVs MCF-7) and 0.51 ± 0.14 (EVs MDA), suggesting polydisperse distributions. The zeta potential of the EVs MCF-7 was -32.6 ± 1.42 mV and EVs MDA was -33.1 ± 1.05 mV, suggesting a high dispersion stability. The spherical morphology of all EVs was confirmed by TEM. The presence of HSP70, CD63 and p53 were demonstrated in the EVs, as well as the presence of amyloid oligomers.

Conclusões

With these results, we expect to improve the understanding of this phenomenon, which could serve as a new pharmacological target, and define a new cancer therapeutic strategy.

Palavras-chave

Mutant p53; Amyloid aggregation; Extracellular vesicles

Financiador do resumo

Área

Estudo Clínico - Tumores de Mama