Abstract
BackgrounduPAR and MMP-9, which play critical roles in tumor cell invasion, migration and angiogenesis, have been shown to be associated with lipid rafts.MethodsTo investigate whether cholesterol could regulate uPAR and MMP-9 in breast carcinoma, we used MβCD (methyl beta cyclodextrin, which extracts cholesterol from lipid rafts) to disrupt lipid rafts and studied its effect on breast cancer cell migration, invasion, angiogenesis and signaling.ResultsMorphological evidence showed the association of uPAR with lipid rafts in breast carcinoma cells. MβCD treatment significantly reduced the colocalization of uPAR and MMP-9 with lipid raft markers and also significantly reduced uPAR and MMP-9 at both the protein and mRNA levels. Spheroid migration and invasion assays showed inhibition of breast carcinoma cell migration and invasion after MβCD treatment. In vitro angiogenesis studies showed a significant decrease in the angiogenic potential of cells pretreated with MβCD. MβCD treatment significantly reduced the levels of MMP-9 and uPAR in raft fractions of MDA-MB-231 and ZR 751 cells. Phosphorylated forms of Src, FAK, Cav, Akt and ERK were significantly inhibited upon MβCD treatment. Increased levels of soluble uPAR were observed upon MβCD treatment. Cholesterol supplementation restored uPAR expression to basal levels in breast carcinoma cell lines. Increased colocalization of uPAR with the lysosomal marker LAMP1 was observed in MβCD-treated cells when compared with untreated cells.ConclusionTaken together, our results suggest that cholesterol levels in lipid rafts are critical for the migration, invasion, and angiogenesis of breast carcinoma cells and could be a critical regulatory factor in these cancer cell processes mediated by uPAR and MMP-9.
Highlights
UPAR and MMP-9, which play critical roles in tumor cell invasion, migration and angiogenesis, have been shown to be associated with lipid rafts
As uPAR and MMP-9 are known to be associated with cholesterol-enriched lipid rafts, the present study investigated the effects of cholesterol depletion-mediated lipid raft disruption by methyl beta cyclodextrin (MbCD) treatment on uPAR and MMP-9 in breast carcinoma cells
The present study shows that lipid raft disruption by cholesterol depletion could significantly attenuate invasion, migration and angiogenesis in breast carcinoma cells
Summary
UPAR and MMP-9, which play critical roles in tumor cell invasion, migration and angiogenesis, have been shown to be associated with lipid rafts. Lipid rafts are detergent-insoluble, cholesterol-rich microdomains and have been attributed to several cellular functions. Lipid rafts play critical roles in the regulation of several membrane receptors, apoptosis, cell adhesion, and protein sorting during endocytosis and exocytosis [1,2]. Cunningham and colleagues [7] have shown that dimerized uPAR partitions preferentially to detergent-resistant lipid rafts. UPAR seems to associate with L-selectin in human neutrophil lipid rafts but not with the complement receptor CR3 [8]. Zhang et al [14] have shown that MMP-9 associates with lipid rafts in highly metastatic sublines from Lewis Lung carcinoma
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