Abstract 5175: L1 stimulation of human glioma cell motility correlates with FAK activation

Abstract

Abstract Our previous studies showed the expression and proteolysis of neural adhesion molecule L1 (CD171) in human glioma cell lines and human glioma surgical samples. The proteolysis of this molecule was suggested to autocrine stimulate glioma cell motility through binding to integrin receptors (Yang et al., 2009). To further investigate the mechanism of this stimulation, in this study, a lentivirally introduced shRNA was used to attenuate L1 expression in human glioma T98G cells (T98G/shL1 cells), which resulted in 95% decreased expression compared to empty vector infected control cells. L1 attenuated cells exhibited 50% decreased cell motility compared to control cells as shown by time lapse microscopy and the quantitative “Super Scratch” assay. These slower T98G/shL1 cells showed a 68% decrease in FAK activity and an approximately 36% increase in focal complex size compared to control cells. These results suggest that T98G/shL1 cells had decreased focal complex turnover, which resulted in their slower migration. Conditioned media containing the L1 ectodomain was made from CHO cells infected with a lentiviral vector expressing the human L1 ectodomain sequence. Adding L1 conditioned media 1) rescued the cell motility in T98G/shL1 cells and 2) increased cell motility in U118 cells (a grade III human glioma cell line lacking of endogenous L1 expression) by 30% compared to the control cells. These results support a model for stimulation of glioma cell motility through the L1 ectodomain after ADAM10 proteolysis (Yang et al., 2009). In order to examine L1 function in glioma cell invasion into brain tissue, a novel embryonic chick model was used. Injection of GFP labeled T98G cells and dissociated cells from human glioma surgical samples into embryonic chick brains resulted in extensive invasion after 4 days, and this in vivo system is being used to study L1 function in glioma cell invasion. Confocal microscopy of immunostained tissue sections revealed that cell surface L1 was missing in cells located at the margin of the tumor as opposed to cells in the tumor mass. These results are consistent with our previous finding that L1 was expressed on the surfaces of confluent T98G cells in culture, but was lost from the cell surface along the edge of a scratch into which cells migrated. These studies suggest a general mechanism where the L1 ectodomain autocrine stimulates glioma cell motility and invasion by binding to integrin receptors, which activates FAK and thereby increases turnover of focal complexes. Supported by NIH INBRE. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5175.