Résumé
Background: Metastasis is the leading cause of breast cancer mortality and is the result of the interplay between cancer cells and the surrounding microenvironment, which is regulated by complex molecular networks and involved numerous genes. Matrix components and many extracellular matrix (ECM) proteins, such as fibronectin (FN), promote tumor progression and metastatic spread. Integrins are cell-surface adhesion receptors that interact with ECM components have been reported to contribute to tumor growth, cell adhesion, invasion, and metastasis.Aim: The purpose of this study was to characterize the role of integrin α5 (ITGA5) and integrin β3 (ITGB3) in cell migration in a model of metastatic breast cancer.
Methods: ITGA5 and ITGB3 expressions were invalidated using siRNA in MDA-MB-231 cells. To achieve a stable knockdown, ITGA5 and ITGB3 invalidation as also performed using shRNA. First, the levels of mRNA and protein were assessed to confirm the effectiveness of the invalidation. Second, impacts of integrin invalidation were evaluated on phenotype characteristics, cytotoxicity, and migratory capacity.
Results: Effective invalidation of ITGA5 and ITGB3 using siRNA, with a strong effect on the migratory capacity in ITGB3 invalidated cells. On the other hand, the ITGA5 knockdown using shRNA was effective in the case of sh126, but it was not for sh124. No differences were observed at the morphological level. A difference in the confluency was observed without a toxicity effect, suggesting a decrease in cell adhesion. During the second part of the study a cell adhesion test was carried out, which did not show conclusive results. Additionally, ITGA5- and ITGB3-invalidated cells were further characterized on uncoated and FN-coated surfaces. A new ITGA5-targeting shRNA was tested, giving a very strong phenotype that did not allow cell characterization. ITGB3 knockdown was performed using shRNA previously tested in the PACMAN project, which covers this study. FN-coating showed an effect on the cells. It triggered the clusterization of ITGA5 and induced change in cell size, indicating a higher adhesion to the surface. Finally, cells were co-invalidated for ITGA5 and ITGB3, leading to a lower adhesion. Nevertheless, the focal adhesion formation was observed in all the cases studied.
Conclusion: ITGA5 invalidated cells did not show a significant difference in their migratory capacity. A further characterization of the role of the integrins in cell migration will allow to identify therapeutic/diagnostic targets against advanced breast cancer.
| la date de réponse | 17 janv. 2019 |
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| langue originale | Anglais |
| L'institution diplômante |
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| Superviseur | Carine Michiels (Promoteur) |