Papel pró-tumoral da trombospondina-1 (TSP-1) em modelos de células de adenocarcinoma mamário

Abstract

Thrombospondin-1 (TSP-1) was described in 1990 as the first physiologic angiogenesis inhibitor, but today its role in tumor angiogenesis and metastasis remains controversial. The N-terminal heparin-binding domain of TSP-1 (NTSP-1) is rather pro-angiogenic, partially through its ability to potentiate the activity of growth factor-dependent receptor tyrosine kinases (RTKs), in endothelial cells. Since NTSP-1 also binds to a vast array of cancer cells, it is important to ascertain if the molecule also affects relevant RTKs in tumor contexts. MCF-10A (hyperplastic), MCF-7 (tumoral; noninvasive) e MDA-MB-231 (tumoral; invasive) breast cell lines were characterized for the expression of NTSP-1 receptors and then investigated for their responses to EGF, since its receptor EGFR was the most consistently detected in all three cell lines. Treatments (NTSP-1 or EGF, alone or in combination) were performed before the following functional assays: (i) wound healing (for assessment of collective migration); (ii) cell tracking (for individual migration); (iii) analysis of cell death and proliferation; (iv) organoid differentiation (3D-assay; except for MDA-MB-231 cells); (v) cell signaling through EGFR and the RasERK pathway. No significant changes in the homeostasis between cell proliferation and death were observed in MCF-10A and MDA-MB-231 cells with the treatments, while NTSP-1/ EGF treatment stimulated MCF-7 cell proliferation. We found significant shifts in individual migration patterns. Among the most important, we observed that MCF-10A e MDA-MB231 cells treated with NTSP-1/EGF exhibited larger accumulated distances, while EGF and NTSP-1/EGF induced significant increases in cell speed, in MCF-7 cells. Overall, for the three lineages, the cell tracking analysis showed that NSTP-1 (alone or in combination with EGF) favored random patterns of cell migration, a trait that rather correlates with the acquisition of metastatic phenotypes. MCF-10A and MCF-7 cell lines grown on a three-dimensional (3D) environment showed both important morphological transitions, but only in MCF-7 cells NTSP-1 enhanced EGF-induced changes in spheroids, from clustered phenotypes to a grape-like and multicellular streaming organization, both considered less differentiated and/or more migratory. We investigated the canonical pathway Ras - - ERK1/2 upon EGFR activation. NTSP-1 and EGF did not appear to act synergistically in the activation of EGFR and ERK1/ 2 in MCF-10A and MDA-MB-231 cells. However, a strong trend towards ERK1/2 potentiation by NTSP-1/EGF was observed in MCF-7 cells, including a strong reduction in the unphosphorylated form of EGFR, suggesting an increase in its endocytosis. This was confirmed by confocal microscopy, which demonstrated the persistence of EGFR co-localization with Rab5A (a primary endosome marker) observed for up to 60 minutes only under the NTSP-1/EGF treatment, suggesting that NTSP-1 prolongs EGF action in these cells. The analysis of ERK activation in MCF-7 cells, when NTSP-1 was replaced by TSP Hep II - a syndecan-4-binding bioactive peptide - showed a tendency for increased activation. In clonogenic assays, treatment with TSP Hep II, alone or in combination with EGF, led to the formation of colonies smaller in diameter, with a dispersed cell appearance. Finally, we characterized the potentiation of another growth factor, FGF-2, in the course of FGFR1-dependent angiogenic response in microvascular ECs. Our data support the hypothesis of a pro-tumor and pro-angiogenic role for the TSP-1 N-terminal domain in the breast microenvironment, in synergy with growth factors