| dc.description.abstract | Autophagy and apoptosis are two important cellular processes, whose dysfunctions are involved in many severe diseases, including cancer. Bulk autophagy is typically mediated by the sequestration of cytoplasmic material into double-membrane vesicles (autophagosomes), which subsequently fuse with lysosomes to have their content degraded and recycled. Studies in yeast have revealed a critical role of the Atg8 (autophagy-related 8) protein in autophagosome formation as well as for the selective recruitment of cargo. Mammalian cells contain several Atg8 homologues (mAtg8s) that can be divided into the LC3 and GABARAP subfamilies. Whereas LC3 and GABARAP proteins have all been associated with cargo recruitment, their differential roles in autophagosome formation and bulk autophagy have been incompletely characterized in too few functional assays. LC3 is the most studied member of the mAtg8s and is widely used as a marker for autophagy. Apoptosis is a major form of programmed cell death, which can be induced intrinsically or extrinsically by death receptor ligands, such as TRAIL, and is commonly mediated by activation of caspase cascades. Autophagy and autophagy-related proteins, including mAtg8s, have been reported to influence apoptosis at different levels and through diverse mechanisms, to either prevent or contribute to cell death. This study critically investigated the role of mAtg8 proteins in bulk autophagy and their influence on TRAIL-induced cell death in two prostate cancer cell lines (LNCaP and PC3). To that end, we simultaneously depleted either one or both of the mAtg8 protein subfamilies and measured changes in TRAIL-induced cell death and bulk autophagy. Our experiments demonstrated that, when induced by starvation or with an inhibitor of the autophagy suppressor mTOR, bulk autophagy required proteins of the GABARAP subfamily, but not LC3s, in both cell lines. This was most surprising, given the previously assumed role of LC3 in bulk autophagy. Basal bulk autophagy likewise depended on GABARAPs, but not LC3s, in LNCaP cells. Intriguingly, however, none of the mAtg8 subfamilies l were required for basal bulk autophagy in PC3 cells. Similar results were obtained in cells that had been treated with TRAIL under nutrient-rich conditions. TRAIL-resistant LNCaP cells did not alter their cell death response upon depletion of any of the mAtg8s, indicating an autophagy-independent resistance mechanism. Partly TRAIL-sensitive PC3 cells, on the other hand, were sensitised to TRAIL-induced Caspase-8 processing and apoptosis upon depletion of the GABARAP subfamily, but not the LC3 subfamily. Based on our obtained results, we conclude that this sensitising effect is unrelated to inhibition of non-selective bulk autophagy, and that it may rather be related to a GABARAP-dependent form of selective autophagy, and/or to a more direct interaction of GABARAP subfamily members with the TRAIL-induced apoptotic machinery. | eng |