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Rôles de la protéine Damaged-DNA Binding 2 sur l'adhérence, les propriétés nanomécaniques et la voie du TGFβ1 dans les cellules tumorales mammaires

Abstract : Understanding of molecular mechanisms behind metastatic progression remains a major issue in cancer research and requires discovery of new predictive markers. Thus, the laboratory has highlighted that DDB2 protein (Damaged-DNA Binding 2), usually involved in DNA repair, is an important factor in breast tumorigenesis. Indeed DDB2, by regulating genes encoding MnSOD and IκBα, is associated with increased proliferation and reduced invasive abilities of breast tumor cells. Since migratory and invasive properties are closely related to adhesive properties, the aim of this work has been to study the involvement of DDB2 in breast cancer cell adhesion. To this end, two breast cancer cell lines have been used: low-DDB2 expressing MDA-MB231 cells, in which this protein was overexpressed and MCF7, which endogenously express DDB2 in which its expression was inhibited. First, adhesive properties of cells in which DDB2 expression is modulated, has been assessed, and reveal that DDB2 expression is associated with a decrease of adhesion on neutral surfaces, suggesting that membrane properties and contents could be modified in these cells. Thus, an analysis of nanomechanical properties by atomic force microscopy indicates a decrease of cell stiffness in DDB2-expressing cells. These data could be related to the loss of cortical actin cytoskeleton observed by cytoimmunofluorescence. To understand molecular mechanisms involved in DDB2-dependent modulation of these properties, a transcriptomic study on adhesion-related encoding genes has been performed (PCR array). This investigation reveals that the transcript level of genes encoding TGFβ1, CTGF and ICAM-1 are modulated according to DDB2 expression level in the two cell lines. Second, we have studied the influence of DDB2 on the TGFβ signaling pathway. Our results show that DDB2, when it is overexpressed, promotes an inhibition of Smads transcriptional activity and the TGFβ1-induced gene expression. We have attempted to understand how DDB2 interferes with this pathway. Our results show that DDB2 seems to decrease the phosphorylation of Smads. In addition, we show by chromatin immunoprecipitation that DDB2 promotes a strong decrease of Smads on the promoter of TGFβ1-induced PAI1 gene, by binding near their specific response elements. Taken together, these data indicate that the nanomechanical properties of breast tumor cell membranes and the TGFβ1-induced signaling pathway would be modulated by DDB2. Also, the present work confirms the importance of DDB2 in breast tumorigenesis and contributes to better define this protein as a new pronostic and/or predictive marker of metastatic progression of breast cancer.
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https://hal.archives-ouvertes.fr/tel-01571606
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Submitted on : Thursday, August 3, 2017 - 9:26:49 AM
Last modification on : Monday, October 10, 2022 - 11:06:24 AM

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  • HAL Id : tel-01571606, version 2

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Claire Barbieux. Rôles de la protéine Damaged-DNA Binding 2 sur l'adhérence, les propriétés nanomécaniques et la voie du TGFβ1 dans les cellules tumorales mammaires. Automatique / Robotique. Université de Lorraine, 2015. Français. ⟨NNT : 2015LORR0337⟩. ⟨tel-01571606v2⟩

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