The Studies On The Function And Mechanism Of Lipid Ratf In β1Integirn-mediated Melanoma Cell Spreading

Cancer cell migration plays a central role in different stages of cancer metastasis. In vivo,in order to migrate to a new tissue site, cancer cell must cross through the connective tissuebarriers, which requires cell-extracellular matrix (ECM) interaction. In vitro, cell adhesionand spreading in response to ECM is the fundmental event of cell migration and requiresintegrin-mediated cell-ECM interaction. Integrins function through binding to ECM andsubsequent clustering to initiate focal adhesion formation and the physical connectionbetween extracellular matrix and cytoskeleton. However, their functions must be regulated byother factors.Lipid raft is a liquid ordered plasma membrane microdomain, enriched in cholesterol andsphingolipid. By excluding or including some proteins selectively, lipid raft provides aspatiotemporal platform for protein-protein, protein-lipid and lipid-lipid interaction. Thecharacteristics in structure and function place lipid raft at the forefront of biomembraneresearch in recent years, and many researchers have paid a great attention to the mechanismby which lipid raft regulates cell motility. Previous studies also indicate that lipid raft iscrucial for regulating the distribution and function of migration-related cell surface receptor,including integrin. However, the role of lipid raft in integrin-mediated cancer cell adhesionand spreading remains unclear.In this study, we identified that lipid raft integrity was required for β1integrin-mediatedinitial spreading of melanoma A375cells on fibronectin. Our results from flow cytometryshowed that A375cells expressed β3and β1integrins abundantly on their surfaces. Byadhesion and spreading assays, we found that β3and β1integrins played important roles inA375cell adhesion and spreading on fibronectin respectively, and disrupting lipid raft byMβCD inhibited β1integrin-mediated A375cell spreading, but not β3integrin-mediatedA375cell adhesion. By immunofluorescence double-labeling experiments, we found that β1integrin clustering depended on intact lipid raft, disruption of which led to the inability of β1integrin-mediated focal adhesion formation and actin cytoskeleton rearrangement.Furthermore, by co-immunoprecipitation and lipid raft fraction isolation, we explored the possible mechanism by which lipid raft regulates β1integrin clustering and demonstrated thatintact lipid raft could recruit and modify some adaptor proteins, such as talin, α-actinin,vinculin, paxillin and FAK. Lipid raft could regulate the location of these proteins in lipid raftfractions and facilitate their binding to β1integrin, which may be crucial for β1integrinclustering. Finally, we showed that lipid raft disruption markedly impaired A375cellmigration in both transwell and wound healing models. This study reveals that disruption oflipid raft might be a helpful strategy for prevention, cure and better management of β1integrin-mediated malignant tumors metastasis.