Abstract 169: Inhibition of focal adhesion kinase suppresses gastrin-releasing peptide receptor-mediated liver metastasis in neuroblastoma

Abstract

Abstract We have previously reported that high expression of gastrin-releasing peptide receptors (GRP-R), a member of the G-protein coupled receptor family, is observed in more undifferentiated human neuroblastoma. We have also found that GRP-R-mediated cell signaling plays a critical role in neuroblastoma cell transformation; however, its molecular mechanisms are unknown. Here, we sought to determine the role of focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase, in GRP-R-mediated neuroblastoma cellular transformation. METHODS. Using human neuroblastoma tissue sections, the correlation of GRP-R and FAK expression was examined. Human neuroblastoma cell line, BE(2)-C and SK-N-SH were used for our experiments. Using transfection with Lipofectamine 2000, the level of knockdown and overexpression of GRP-R and FAK were modulated. MTT assay was carried out to measure the cell viability of neuroblastoma cells, and soft agar colony formation assay was performed to determine the capacity of cell transformation. Transwell migration and wound healing assay were employed to study cell motility. Rescue experiments by overexpression of FAK in GRP-R silencing cells were conducted to further validate the role of FAK in neuroblastoma cell transformation. Using FAK inhibitor and GRP-R agonist, animal experiment was performed. RESULTS. FAK expression correlated with GRP-R expression in BE(2)-C and SK-N-SH human neuroblastoma cells. GRP treatment induced phosphorylation of FAK at Y397 in both cell lines. Overexpression of GRP-R in SK-N-SH cells, which are known to express low endogenous levels of GRP-R, increased the levels of phosphorylated and total FAK. Moreover, overexpression of FAK increased cell viability and colony formation of SK-N-SH cells. These effects were reversed when cells were co-transfected with FAK siRNA in BE(2)-C cells, which endogenously express higher levels of GRP-R. Conversely, silencing of GRP-R decreased the levels of phosphorylated and total FAK in BE(2)-C cells. Interestingly, in cells with GRP-R silencing, FAK overexpression significantly enhanced cell viability and colony formation. GRP-R-mediated liver metastasis suppressed by FAK inhibitor in vivo. CONCLUSIONS. Our results suggest that FAK is a critical downstream regulator of GRP-R-mediated tumorigenesis. Furthermore, GRP-R and FAK expression correlate with morphological changes implicated in the malignant transformation of human neuroblastoma cells. Importantly, inhibition of FAK can suppress GRP-R-induced tumor progression in neuroblastoma in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 169. doi:1538-7445.AM2012-169