title: HAMLET binding to α-actinin facilitates tumor cell detachment.
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.
authors.affiliation: Institute of Laboratory Medicine, Department of Microbiology, Immunology and Glycobiology, Lund University, Lund, Sweden.
authors.forename: Maria
authors.initials: M
authors.lastname: Trulsson
authors.forename: Hao
authors.initials: H
authors.lastname: Yu
authors.forename: Lennart
authors.initials: L
authors.lastname: Gisselsson
authors.forename: Yinxia
authors.initials: Y
authors.lastname: Chao
authors.forename: Alexander
authors.initials: A
authors.lastname: Urbano
authors.forename: Sonja
authors.initials: S
authors.lastname: Aits
authors.forename: Ann-Kristin
authors.initials: AK
authors.lastname: Mossberg
authors.forename: Catharina
authors.initials: C
authors.lastname: Svanborg
mesh_terms: D000185:Actinin / Q000737:chemistry / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002448:Cell Adhesion / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002457:Cell Extracts; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism; D007768:Lactalbumin / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology*; D009829:Oleic Acids / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D025941:Protein Interaction Mapping; D021381:Protein Transport / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D000199:Actins; D002457:Cell Extracts; C506422:HAMLET complex, human; D019012:Integrin beta1; D009829:Oleic Acids; D010455:Peptides; D000185:Actinin; D007768:Lactalbumin; D051416:Focal Adhesion Protein-Tyrosine Kinases