Abstract LB-193: TRAIL promotes entosis through Caspase-8

Despite its apoptosis-inducing activity, tumor necrosis factor-related apoptosis inducing ligand (TRAIL) also activates survival signaling pathways. Survival signaling not only interferes with apoptosis and causes resistance, but can also lead to gain of several tumor-promoting functions such as proliferation, invasion and metastasis. Here we demonstrate that colorectal cancer cells surviving from TRAIL treatment can initiate a cell-in-cell invasion process known as entosis. Entosis refers to a unique cell-in-cell phenomenon, in which one or more viable cells are actively internalized into another cell of the same type. After formation of cell-in-cell structures (CICs), internalized cells can divide inside the host cell or they can escape, however, the vast majority of cells undergo cell death inside their hosts, through a lysosome-dependent mechanism. Although the clinical relevance of entosis is highly context-dependent, the frequency of entosis has been shown associated with tumor grade and poor outcome in several malignancies. While monitoring single-cell response upon TRAIL stimulation, we noted that some cells internalized into their neighboring cells and died inside a vacuole. We confirmed CICs by fluorescence staining of cytoplasm, nucleus, lysosome and cell membrane, in addition to TEM. Further quantification revealed that TRAIL indeed increased the number of CICs in colorectal cancer cell lines (HCT116 and LS180) and spheroids. Moreover, both pre-treatment with ROCK inhibitor (Y-27632) and knockout of Caspase-8 resulted in a significant reduction in the number of TRAIL-induced CICs. After forming cell-in-cell structures, the majority of internalized cells appeared positive for lysotracker staining and underwent cell death inside their hosts. These cells showed an increase in IETD FRET probe cleavage, additionally, we observed increased levels of cleaved caspase-8, cleaved caspase-9 and cleaved caspase-3 in these cells in presence of TRAIL. Pre-treatment with z-VAD-fmk reduced the number of internalized cells undergoing cell death and Bax/Bak double knockout cells escaped from their hosts without undergoing cell death in response to TRAIL. Our findings demonstrate that entosis can be induced by TRAIL in a caspase-8-dependent manner, and may give new insights in understanding the death, survival and escape mechanisms in internalized cells. Citation Format: Emir Bozkurt, Heiko Dussmann, Jochen H. Prehn. TRAIL promotes entosis through Caspase-8 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-193.