Proteomic profiling of proteins associated with urokinase plasminogen activator receptor in a colon cancer cell line using an antisense approach.
Expression of urokinase plasminogen activator (uPA) and its receptor (uPAR) strongly correlates with a malignant tumour cell phenotype. In the multistep process of metastasis, uPA binding to uPAR influences different cellular functions. In the present study, a highly metastatic colon cancer cell line, HCT116 was transfected with an expression vector containing a 5' uPAR cDNA fragment in an antisense orientation. This construct was most effective in reducing uPAR cell surface expression as confirmed by flow cytometry analysis. Antisense transfection of HCT116 cells had no effect on proliferation but the following effects were observed: (1) a 1.3-fold decreased adhesion; (2) a two-fold decreased Erk MAP kinase activity; (3) a 2.7-fold decrease in Src kinase activity; (4) a 1.5- and two-fold decrease in uPA cell surface expression and secretion; (5) abrogation of promatrix metalloproteinase-9 secretion; and (6) a complete suppression of plasminogen-dependent matrix degradation. Using proteomic analysis, we demonstrate loss of approximately 200 proteins and quantitative differences in the expression of 141 other proteins in an antisense-clone compared to wild-type and mock-transfected control. Such changes in protein expression with the down-regulation of uPAR may be an important contributor in colon cancer progression and metastasis and may not only provide a basis to develop a proteomic data bank of uPAR-mediated signaling molecules but may also lead to the development of therapeutic approaches for the cure and better management of colon cancer.
Ahmed
Nuzhat
N
Gynaecological Cancer Research Centre, Royal Women's Hospital, University of Melbourne, Australia. nuzhata@unimelb.edu.au
Oliva
Karen
K
Wang
Yao
Y
Quinn
Michael
M
Rice
Greg
G
D020121:5' Untranslated Regions; D001485:Basement Membrane / Q000378:metabolism; D015153:Blotting, Western; D002448:Cell Adhesion; D002455:Cell Division; D045744:Cell Line, Tumor; D024141:Collagen Type IV / Q000737:chemistry; D003110:Colonic Neoplasms / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D015180:Electrophoresis, Gel, Two-Dimensional; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D020935:MAP Kinase Signaling System; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D009362:Neoplasm Metastasis; D016376:Oligonucleotides, Antisense / Q000378:metabolism*; D010641:Phenotype; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D015398:Signal Transduction; D014162:Transfection; D019061:src-Family Kinases / Q000378:metabolism
D020121:5' Untranslated Regions; D024141:Collagen Type IV; D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D016376:Oligonucleotides, Antisense; C524898:PLAUR protein, human; D020543:Proteome; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D019061:src-Family Kinases; D020780:Matrix Metalloproteinase 9