title: Localization of uPAR and MMP-9 in lipid rafts is critical for migration, invasion and angiogenesis in human breast cancer cells.
abstract: BACKGROUND
  uPAR and MMP-9, which play critical roles in tumor cell invasion, migration and angiogenesis, have been shown to be associated with lipid rafts.
  
  
  METHODS
  To 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.
  
  
  RESULTS
  Morphological 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.
  
  
  CONCLUSIONS
  Taken 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.
authors.affiliation: Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, One Illini Drive, Peoria, IL 61605, USA.
authors.forename: Hari
authors.initials: H
authors.lastname: Raghu
authors.forename: Prasanna Kumar
authors.initials: PK
authors.lastname: Sodadasu
authors.forename: Rama Rao
authors.initials: RR
authors.lastname: Malla
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Gondi
authors.forename: Norman
authors.initials: N
authors.lastname: Estes
authors.forename: Jasti S
authors.initials: JS
authors.lastname: Rao
mesh_terms: D001943:Breast Neoplasms / Q000098:blood supply / Q000201:enzymology* / Q000473:pathology; D022461:Caveolins / Q000378:metabolism; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D002784:Cholesterol / Q000172:deficiency; D017077:Culture Media, Conditioned / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000201:enzymology* / Q000473:pathology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D021962:Membrane Microdomains / Q000187:drug effects / Q000201:enzymology*; D009361:Neoplasm Invasiveness; D009389:Neovascularization, Pathologic / Q000201:enzymology* / Q000473:pathology; D009761:Nystatin / Q000494:pharmacology; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D047392:beta-Cyclodextrins / Q000494:pharmacology; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D022461:Caveolins; D017077:Culture Media, Conditioned; C524898:PLAUR protein, human; D012333:RNA, Messenger; D055293:Receptors, Urokinase Plasminogen Activator; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D009761:Nystatin; D002784:Cholesterol; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; D019061:src-Family Kinases; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D020780:Matrix Metalloproteinase 9