2D-DIGE analysis of phospho-enriched fractions from dasatinib-treated melanoma cell lines.
Current therapeutic regimes for metastatic melanoma have failed to provide robust clinical responses. Dasatinib has shown anti-proliferative and anti-invasive effects in vitro; however, not all melanoma cells tested were sensitive to dasatinib. We used 2D-DIGE analysis of phospho-enriched fractions to identify phosphoproteins involved in regulating response to dasatinib in an isogenic pair of melanoma cell lines, one sensitive to dasatinib (WM-115) and the other resistant (WM-266-4). In WM-115 cells treated with dasatinib, 18 unique protein species with altered phosphorylation levels were detected. Dasatinib treatment of WM-266-4 cells resulted in phosphoprotein alterations to four unique protein species. Four phosphorylated forms of Annexin-A2 (ANXA2) were increased in WM-115 cells treated with dasatinib, whilst dasatinib treatment did not alter ANXA2 phosphoprotein levels in WM-266-4 cells. Immunoblotting confirmed that phosphorylation of ANXA2, on tyrosine residues, was increased in WM-115 cells treated with dasatinib. Subsequent knockdown of ANXA2 by siRNA significantly inhibited proliferation of WM-115 cells but did not significantly reduce proliferation of WM-266-4 cells. Therefore, ANXA2 plays a role in regulating proliferation in dasatinib-sensitive WM-115 cells and could potentially play a role in sensitivity to dasatinib in melanoma cells.
National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, Ireland. Alex.Eustace@dcu.ie
Alex J
AJ
Eustace
Paul
P
Dowling
Michael
M
Henry
Padraig
P
Doolan
Paula
P
Meleady
Martin
M
Clynes
John
J
Crown
Norma
N
O'Donovan
D000595:Amino Acid Sequence; D017306:Annexin A2 / Q000378:metabolism / Q000502:physiology; D002458:Cell Fractionation; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D000069439:Dasatinib; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D006801:Humans; D008545:Melanoma / Q000378:metabolism / Q000473:pathology*; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D020543:Proteome / Q000032:analysis / Q000187:drug effects; D011743:Pyrimidines / Q000494:pharmacology*; D013844:Thiazoles / Q000494:pharmacology*; D057825:Two-Dimensional Difference Gel Electrophoresis / Q000379:methods
C472822:ANXA2 protein, human; D017306:Annexin A2; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D020543:Proteome; D011743:Pyrimidines; D013844:Thiazoles; D000069439:Dasatinib