STAT5 regulates glioma cell invasion by pathways dependent and independent of STAT5 DNA binding

Abstract

STAT5 activation in primary glioma was analyzed using antibody directed against phosphorylated STAT5 (pSTAT5), in all samples robust pSTAT5 immunostaining was detected, predominantly in the nucleus. We then used immunofluorescence, transcription factor binding assays and western blotting to study EGF-modulated STAT5 activation in the human U87 glioma cell line. EGF was found to upregulate pSTAT5 levels and enhances STAT5 DNA-binding activity. To address the role of STAT5 in glioma cell invasion, resting and EGF-treated U87 cells were treated with siRNA directed against STAT5 and the extent of glioma cell migration into a Matrigel matrix was monitored. EGF treatment markedly enhanced matrix invasion, and knockdown of STAT5 expression with siRNA significantly downregulated matrix invasion by both EGF-stimulated and resting glioma cells. However, transfection with a decoy oligonucleotide abolished transcriptional activation of a STAT5 target gene but failed to inhibit matrix invasion in resting U87 cells. By contrast, the decoy nucleotide abolished EGF enhancement of matrix invasion. STAT5 therefore promotes glioma cell invasion via at least two different pathways: a pathway dependent of STAT5 DNA binding, and a second pathway independent of STAT5 DNA binding.