HAMLET Binding to α-Actinin Facilitates Tumor Cell Detachment

Cell adhesion is tightly regulated by specific molecular interactions and detachment from the extracellular matrix modifies proliferation and survival. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a protein-lipid complex with tumoricidal activity that also triggers tumor cell detachment in vitro and in vivo, suggesting that molecular interactions defining detachment are perturbed in cancer cells. To identify such interactions, cell membrane extracts were used in Far-western blots and HAMLET was shown to bind alpha-actinins; major F-actin cross-linking proteins and focal adhesion constituents. Synthetic peptide mapping revealed that HAMLET binds to the N-terminal actin-binding domain as well as the integrin-binding domain of alpha-actinin-4. By co-immunoprecipitation of extracts from HAMLET-treated cancer cells, an interaction with alpha-actinin-1 and -4 was observed. Inhibition of alpha-actinin-1 and alpha-actinin-4 expression by siRNA transfection increased detachment, while alpha-actinin-4-GFP over-expression significantly delayed rounding up and detachment of tumor cells in response to HAMLET. In response to HAMLET, adherent tumor cells rounded up and detached, suggesting a loss of the actin cytoskeletal organization. These changes were accompanied by a reduction in beta 1 integrin staining and a decrease in FAK and ERK1/2 phosphorylation, consistent with a disruption of integrin-dependent cell adhesion signaling. Detachment per se did not increase cell death during the 22 hour experimental period, regardless of alpha-actinin-4 and alpha-actinin-1 expression levels but adherent cells with low alpha-actinin levels showed increased death in response to HAMLET. The results suggest that the interaction between HAMLET and alpha-actinins promotes tumor cell detachment. As alpha-actinins also associate with signaling molecules, cytoplasmic domains of transmembrane receptors and ion channels, additional alpha-actinin-dependent mechanisms are discussed.