Thèse de doctorat en Biologie cellulaire et moléculaire
Sous la direction de Jacques Pouysségur.
Soutenue en 2008
à Nice , dans le cadre de École doctorale des Sciences de la vie et de la santé (Sophia Antipolis, Alpes-Maritimes) .
Signalisation hypoxique : le rôle de BNIP3 dans la prolifération et l'autophagy
Pas de résumé disponible.
Pas de résumé disponible.
As solid tumors grow the vascular network becomes insufficient for nutrient delivery and oxygen demand. The cells’ proximity to existing blood vessels leads to tumors characterized by areas of high oxygen tension interspersed with areas of relative hypoxia. Tumor cells respond by inducing and stabilizing the hypoxia-inducible factor 1 (HIF-1). HIF-1 regulates a complex gene expression network involved in the induction of angiogenesis, anaerobic metabolism, pHi regulation, migration and invasion, but also by decreasing tumor proliferation. The pathophysiological consequences and underlying molecular mechanisms of the HIF-dependent adaptation to hypoxia and HIF-dependent gene expression are far for being fully characterized. Two of these genes are the hypoxia-inducible pro-apoptotic molecule, Bcl-2/adenovirus E1B 19 KDa interacting protein 3 (BNIP3) and Bcl2/adenovirus E1B 19 kDa interacting protein 3-Like (BNIPL). BNIP3 and BNIP3L are part of the BH3-only Bcl-2 family. The members of this subgroup are characterized as pro-apoptotic and subsequently many investigations have focused on the role of BNIP3 in apoptosis. Some researchers have identified BNIP3 as a positive regulator of autophagy by promoting autophagosome formation and have suggested that this might be the mechanism by which BNIP3 promotes cell death. Nevertheless, many groups have been unable to see an impact of BNIP3 on cell death. In this study, we investigated the role of BNIP3 in normal and cancerous cells under hypoxic conditions in vitro and in vivo. We report that hypoxia-induced BNIP3 decreases colon carcinoma tumor proliferation as much as 70% in mice. Extracts from BNIP3-silenced tumors displayed a significantly higher level in ERK phosphorylation than control tumors. Hypoxia-induced or forced over-expression of BNIP3 in cultured hamster fibroblast (CCL39) and human colon carcinoma cells (BE) resulted in a significant decreased in active ERK, accumulation of the cdk inhibitor p27 with a consequent increase in the length of G1-phase and a significant decrease in cell proliferation. Conversely, knocking down of BNIP3 expression was sufficient to prevent the hypoxia-induced decrease in ERK activity and maintain normal cell proliferation. The BNIP3-dependent decrease in ERK activation was associated with increased MAPK phosphatase activity in vitro and higher dual specificity phosphatases (DUSPs) mRNAs in vivo. These studies establish the physiological significance of BNIP3 activity and begin to delineate a hypoxia-induced BNIP3 signaling pathway that acts through ERK regulation. Furthermore, we have been able to identify HIF-1-induced BNIP3L, which together with BNIP3, activates the autophagy process. As BNIP3L induction is observed to more extreme hypoxic conditions (0. 1%) it might relay the action of BNIP3 induced as early as 3% oxygen. First, while siRNA mediated ablation of either BNIP3 or BNIP3L has little effect on autophagy, combined silencing of these two HIF targets suppressed hypoxia-mediated autophagy. Second, ectopic expression of both BNIP3 and BNIP3L in normoxia activates autophagy. Third, 20-mer BH3-peptides of BNIP3 or BNIP3L were found to be sufficient to initiate autophagy in normoxia. Thus we propose a model in which the atypical BH3-domains of hypoxia-induced BNIP3/BNIP3L have been ‘designed’ to induce autophagy by disrupting the Beclin1-Bcl-2 complex without inducing cell death. Based on these results, we propose that hypoxia-induced BNIP3 participates in the general HIF-induced adaptive mechanism leading to tumor cell survival through the attenuation of the rate of cell proliferation decreasing ATP demand but also through the activation and regulation of autophagy in response to nutriment availability.