Biochemical and Molecular Characterisation of the FADDosome

An anti-metabolite drug, 5-FU (5-fluorouracil), is widely used in the treatment of colorectal cancer. However, acquisition of resistance by cancer cells is the main cause of the limited clinical use of 5-FU, which needs to be overcome to widen and lengthen its utility. Therefore, it is necessary to investigate the factors and pathways involved in 5-FU-induced apoptosis. Chemotherapeutic agents, such as 5-FU, are believed to exert their anti-cancer effect by initiation of apoptosis via the intrinsic cell death pathway, which requires activation of caspase-9. However, our group has recently discovered that 5-FU-induced cell death is initiated by activation of caspase-8, whereas caspase-9 was found to be dispensable for this process. Moreover, our group has found that upon treatment with 5-FU, caspase-10 is upregulated, which leads to the formation of a newly discovered apoptosis-inducing protein complex, FADDosome. This study is composed of two parts. In part I, the functional and interactional analysis of the main FADDosome constituents revealed that caspase-10 has the ability to activate caspase-8 in vitro. Moreover, it was found that FADD and caspase-8 possibly interact via a novel, DED-independent mechanism in vitro. In part II, our group discovered that the ATR, Chk1 and Chk2 kinases are activated in 5-FU-treated HCT116 cells and play a key role in the 5-FU-induced DNA damage signal transduction towards caspase-10 upregulation and FADDosome formation. It was found that activation of Chk1 and Chk2 as well as induction of p53 are regulated in an ATR-dependent manner in response to 5-FU. It was discovered that when Chk1 and Chk2 are inhibited, the 5-FU-treated HCT116 cells appear to shift to an alternative cell death mechanism, mediated by another newly discovered apoptosis-inducing complex, the FLIPosome. The obtained results provide new insights into the roles of the proteins and mechanism involved in the FADDosome formation and apoptosis regulation in colorectal cancer cells treated with 5-FU.