Inhibition of phospholipase C-gamma 1 activation blocks glioma cell motility and invasion of fetal rat brain aggregates

OBJECTIVE: Phospholipase C (PLC)-gamma is a cytosolic enzyme activated by s everal growth factor (GF) receptors (epidermal GF receptor [EGFR], platelet -derived GF receptor, and insulin-like GF 1 receptor), and its activation i s associated with increased cell motility (but not cell proliferation) in n onglioma cell lines. Because up-regulated activation of EGFR has been consi stently linked to poor patient survival in patients with glioblastoma multi forme (GBM) and because inhibition of EGFR activation by tyrosine kinase in hibitors prevents glioma infiltration in vitro, we hypothesized that inhibi tion of PLC-gamma activation would inhibit glioma cell invasiveness. METHODS: Our experimental model assesses tumor spheroid invasion of fetal r at brain spheroids by confocal microscopy. We treated U87 GEM spheroids, an d those derived from a single patient, with the PLC inhibitor U73122. We al so transfected rat C6 glioma cells with the PLCz complementary deoxyribonuc leic acid coding for a dominant negative PLC-gamma 1 src-homology-2/src-hom ology-3 peptide fragment, which blocks binding and activation of PLC-gamma 1 by GF receptors. Two clones (C6F and C6E) were grown into spheroids and w ere tested for invasiveness in the spheroid model and for responsiveness to GFs in a standard in vitro motility assay. RESULTS: The infiltration rate of the patient GBM cell line overexpressing wild-type EGFR was reduced by 2 mu mol/L U73122 from a slope (percent invas ion/h) of 0.74 +/- 0.08 (with the inactive congener U73343) to 0.04 +/- 0.0 53 (P = 8 x 10(-7) by two-tailed t test, 92% reduction); the integral rate, another measure of invasion, was reduced from 49.7 +/- 13 percent-hours pe r hour to 13.6 +/- 12 (P = 0.002, 72% reduction). The U87 spheroid invasion rate was reduced by 0.5 mu mol/L U73122 from 46.7 +/- 8.5 percent-hours pe r hour to 11.2 +/- 4.6 (P = 3 x 10(-5)); th, slope decreased from 1.7 +/- 0 .41 percent per hour to 0.35 +/- 0.14 (P = 0.0001). The C6F and C6E clones demonstrated attachment to and "surrounding" of the fetal rat brain aggrega te but no true invasion by confocal or light microscopy. PLCz blocked the m otility response to epidermal CF, platelet-derived GF, and insulin-like CF. There was a significant decrease in PLC-gamma 1-associated tyrosine phosph orylation. CONCLUSION: These results support a key role for PLC-gamma activation as a common postreceptor pathway for GF-induced tumor infiltration and further i dentify PLC-gamma 1 as a possible target for anti-invasive therapy for GBMs .