Gastrin-releasing peptide-mediated neuroblastoma growth: A role for the PI3K/Akt pathway

Neuroblastoma is the most common extracranial solid tumor in infants and children. Our lab and others have shown trophic actions of gastrin-releasing peptide (GRP), and its analogue bombesin (BBS), in neuroblastomas. Our lab also found that undifferentiated neuroblastomas express increased levels of GRP receptor (GRPR). Activation of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway, a crucial regulator of cell survival, is associated with poor outcome in neuroblastomas and our lab¡¦s previous work has shown that GRPR regulates the expression of PI3K/Akt pathway components. However, the signaling mechanisms involved in this process are not clearly defined. Therefore, the objective of this project was to determine how GRP/GRPR, by way of PI3K pathway, regulates neuroblastoma growth. \r\n\r\nGRP/BBS treatment rapidly increased phosphorylation of both Akt and GSK-3ƒÒ in neuroblastoma cells. Antagonism or silencing of GRPR attenuated BBS-induced phosphorylation of Akt. PI3K inhibition also abrogated BBS-stimulated phosphorylated (p)-Akt as well as its cell cycle targets. GRP increased G1/S phase progression in SK-N-SH cells and BBS-mediated BrdU incorporation was blocked with a PI3K inhibitor. These findings identify PI3K/Akt as an important signaling pathway for GRP-mediated neuroblastoma cell growth. In order to determine the in vivo significance of GRP/GRPR, the effects of BBS treatment in nude mice with human neuroblastoma xenografts were assessed. BBS treatment significantly increased the growth and mediators of angiogenesis of SK-N-SH and BE(2)-C tumors, as well as increased p-Akt levels. A GRPR antagonist reduced BBS-stimulated tumor growth and angiogenic markers in vivo. GRP or GRPR silencing inhibited the expressions of VEGF, p-Akt, and p-mTOR in vitro. GRPR knockdown induced cell morphology changes, reduced cell size, decreased cell proliferation, and inhibited DNA synthesis, which corresponded to G2/M cell cycle arrest. Activated Akt and its downstream regulators of protein synthesis and metabolism were also significantly downregulated by GRPR silencing. GRPR knockdown upregulated the expression of PTEN, the inhibitor of the PI3K/Akt pathway. Furthermore, silencing of GRPR or GRP suppressed anchorage-independent growth; while GRPR overexpression resulted in soft agar colony formation, which was inhibited by a GRP-blocking antibody. In conclusion, these findings demonstrate that GRP/GRPR signaling regulates the PI3K/Akt pathway and promotes neuroblastoma growth, angiogenesis, and oncogenic properties.