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| Author(s): |
Suély Vieira da Silva
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) |
| Defense date: | 2019-08-30 |
| Examining board members: |
Vanessa Morais Freitas;
Carolina Beltrame Del Debbio;
Ana Paula Zen Petisco Fiore;
Maria Aparecida da Silva Pinhal
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| Advisor: | Vanessa Morais Freitas |
| Abstract | |
Tumorigenesis is a complex and dynamic process with participation of molecules present in the extracellular matrix for progression. Thus, metalloproteases present in the tumor stroma contribute to tumor invasion, because they act degrading the extracellular matrix structures and increase the malignant potential of cells. ADAMTSs (a disintegrin and metalloprotease with thrombospondin domains) are secreted proteases to the extracellular matrix, which have proteolytic activity on the extracellular matrix components remodeling it. They are known to be extracellular proteases present in the matrix, but we identified one of the members of this family of ADAMTSs, ADAMTS-1, present predominantly in the nucleus of mammary cells with different degrees of differentiation. Thus, the objective of this study was to analyze if ADAMTS-1 would be the only protease of this family located in the nucleus, and what would be the function of this nuclear protease (if it would have enzymatic function). In addition, we attempted to understand how it reaches the nucleus of the cells by analyzing the role of the cytoskeleton in this transport, in addition to assessing whether ADAMTS-1 would be internalized after secreted, or whether it would reach the nucleus through the nucleocytoplasmic transport as described in the literature. Our results demonstrated that ADAMTS-1 is the only aggrecanase present in the nucleus of cells, and when it was silenced, had lower protein expression in this compartment, as well as in the cytoplasm and conditioned medium. ADAMTS-1 nuclear was not observed in all cell lines. We observed that cells with epithelial phenotype exhibit nuclear protease, whereas cells with mesenchymal origin (such as fibroblasts) presented the protease in the cytoplasmic compartment. In an assay of digestion of aggrecan we observed as well as in the extracellular matrix, nuclear ADAMTS-1 also presented enzymatic activity, event observed by the presence of aggrecan fragments generated by proteases in the nuclear fraction of cells. We observed the treatment with monensin impaired the secretion of ADAMTS-1, and accumulation of this 22 protease at the cytoplasmic compartment. Cells of mesenchymal origin after being maintained under medium containing the soluble protease, presented nuclear compartmentalization. Thus, we established a possible role of nuclear ADAMTS-1, with active proteolytic function, since it was the only aggrecanase characterized in the nuclear compartment. We also observed the compartmentalization of protease could be altered if the secretory pathways are disturbed. Using the media secreted by cells with nuclear ADAMTS-1, we have seen that HT-1080 cells and fibroblasts (which do not present nuclear protease) was able to internalize the protease, and the localization of ADAMTS-1 was seen at nuclear compartment. Using the same conditioned medium, we observed which a higher availability of ADAMTS-1 in the extracellular environment decreased the migratory potential of cells with mesenchymal phenotype. We have that ADAMTS-1 is a molecule secreted by cells into the extracellular matrix, which presents the particularity of being endocytosed after being secreted, and its location becomes nuclear. ADAMTS-1 has nuclear localization in cells of epithelial origin, contrary to what is observed in cells of mesenchymal origin. Nuclear ADAMTS-1 may represent an important molecule of the tumor microenvironment, since it has functional proteolytic activity in this compartment. (AU) | |
| FAPESP's process: | 15/09845-9 - Role of ADAMTS-1 (a Disintegrin and Metalloproteinase with Thrombospondin Motifs 1) in nucleus of normal and tumoral human breast cells |
| Grantee: | Suély Vieira da Silva |
| Support Opportunities: | Scholarships in Brazil - Doctorate |