HAMLET binding to α-actinin facilitates tumor cell detachment

• Published in: PloS one 2011-03, Vol.6 (3), p.e17179
• Main Authors: Trulsson, Maria, Yu, Hao, Gisselsson, Lennart, Chao, Yinxia, Urbano, Alexander, Aits, Sonja, Mossberg, Ann-Kristin, Svanborg, Catharina
• Format: Article
• Language: English
• Subjects: Actin; Actinin - chemistry; Actinin - metabolism; Actins - metabolism; Adherent cells; Adhesion; Amino Acid Sequence; Apoptosis; Basic Medicine; Binding; Binding Sites; Biology; Bladder cancer; Cancer; Cell adhesion; Cell adhesion & migration; Cell Adhesion - drug effects; Cell death; Cell Death - drug effects; Cell Extracts; Cell Line, Tumor; Cell proliferation; Cell survival; Cell Survival - drug effects; Chromatography; Crosslinking; Cytoskeleton; Detaching; Detachment; Disruption; Extracellular matrix; Focal adhesion kinase; Focal Adhesion Protein-Tyrosine Kinases - metabolism; Humans; Immunology; Immunoprecipitation; Integrin beta1 - metabolism; Ion channels; Kinases; Laboratories; Lactalbumin; Lactalbumin - metabolism; Medical and Health Sciences; Medicin och hälsovetenskap; Medicine; Medicinska och farmaceutiska grundvetenskaper; Metastasis; Microbiology in the medical area; Mikrobiologi inom det medicinska området; Models, Biological; Models, Molecular; Molecular interactions; Molecular Sequence Data; Morphology; Neoplasms - enzymology; Neoplasms - metabolism; Neoplasms - pathology; Neuroblastoma; Oleic Acids - metabolism; Overexpression; Peptide mapping; Peptides - chemistry; Peptides - metabolism; Peptides - pharmacology; Phosphorylation; Prostate cancer; Protein Binding - drug effects; Protein Interaction Mapping; Protein Transport - drug effects; Proteins; Receptors; Rounding; Signal Transduction - drug effects; Signaling; siRNA; Transfection; Tumor cells; Western blotting; α-Actinin
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0017179

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.