Abstract
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 α-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 α-actinin-4. By co-immunoprecipitation of extracts from HAMLET-treated cancer cells, an interaction with α-actinin-1 and -4 was observed. Inhibition of α-actinin-1 and α-actinin-4 expression by siRNA transfection increased detachment, while α-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 β1 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 α-actinin-4 and α-actinin-1 expression levels but adherent cells with low α-actinin levels showed increased death in response to HAMLET. The results suggest that the interaction between HAMLET and α-actinins promotes tumor cell detachment. As α-actinins also associate with signaling molecules, cytoplasmic domains of transmembrane receptors and ion channels, additional α-actinin-dependent mechanisms are discussed.
Highlights
Cell adhesion is essential for tissue integrity and processes that modify adhesion are tightly regulated [1]
Four human a-actinin isoforms have been described. a-Actinin-1 is found in focal adhesions and various F-actin-based structures [8,9,10]. a-Actinin2 and a-actinin-3 are expressed in cardiac and/or skeletal muscles and cross-link F-actin in the region of Z-discs of muscle cells [11]. a-Actinin-4, which displays 87% sequence identity to a-actinin-1 [9], is detected at points of cell-cell contact [12] and interacts with focal adhesion constituents, including vinculin and the cytoplasmic domain of b integrins [3,6,10]. a-Actinin has been proposed to play a crucial role in the step of de-adhesion e.g. by recruiting MEKK1 and calpains which cleave several focal adhesion proteins
HAMLET binds a-actinin-4 in cell extracts To identify cellular targets involved in detachment, membraneassociated proteins were isolated from A549 carcinoma cells
Summary
Cell adhesion is essential for tissue integrity and processes that modify adhesion are tightly regulated [1]. A-Actinin-4, which displays 87% sequence identity to a-actinin-1 [9], is detected at points of cell-cell contact [12] and interacts with focal adhesion constituents, including vinculin and the cytoplasmic domain of b integrins [3,6,10]. It has been shown that inhibition of a-actinin-4 by shRNA decreased cell-matrix adhesion in some astrocytoma cell lines while a-actinin-1 silencing had no effect or even increased the adhesion [15]. In addition to their role as actin cross-linkers, a-actinins associate with signaling molecules, cytoplasmic domains of transmembrane receptors and ion channels, connecting the cytoskeletal scaffold to diverse signaling processes [16]
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