Cellular interactions of HAMLET and their role in cell death

Sammanfattning: HAMLET is a protein-lipid complex that selectively kills tumor cells. In this thesis, we identified new mechanisms, whereby HAMLET initiates and executes tumor cell death. HAMLET targets several cellular compartments ranging from the plasma membrane to the nucleus. HAMLET rapidly binds to the plasma membrane of tumor cells and the resulting changes in membrane shape and lipid composition activate ion channels and rapid ion fluxes (paper I). Through macropinocytosis, large amounts of HAMLET enter tumor cells, thus reaching intracellular targets (paper II). By interacting with alpha-actinins, HAMLET facilitates tumor cell detachment and perturbs FAK signaling (paper III). Furthermore, HAMLET interacts with HK1, a member of the glycolytic machinery, and thereby disrupts tumor cell metabolism (paper IV). HAMLET also triggers ER stress (paper I). This provides a framework for HAMLET’s ability to rapidly kill a wide range of tumor cells and addresses three major HAMLET questions. 1. How are the tumor cells killed? 2. How is HAMLET internalized by tumor cells? 3. Why do normal differentiated cells survive? We show that HAMLET-induced cell death is initiated at the plasma membrane and requires functional ion channels and p38 MAPK signaling. We identify macropinocytosis as a route for HAMLET internalization and distinguish this process from cell death. Finally, we show that the HAMLET sensitivity reflects tumor cell characteristics, such as c-Myc oncogene expression and altered metabolism, as inhibition of glycolysis increased HAMLET sensitivity. Interestingly, HAMLET does not appear to perturb the membranes of normal differentiated cells, further explaining the tumor selectivity.