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Activation of PI3K/AKT pathway correlates with poor prognosis in patients with neuroblastoma. Our previous studies have demonstrated that PI3K/AKT signaling is critical for the oncogenic transformations induced by gastrin-releasing peptide (GRP) and its receptor, GRP-R, in neuroblastoma. Moreover, PI3K/AKT-dependent oncogenic transformations require N-myc, an extensively studied oncogene in neuroblastoma. Whether AKT directly regulates the expression of N-myc oncogene is yet to be determined. Here, we report a novel finding that of the three AKT isoforms, AKT2 specifically regulated N-myc expression in neuroblastoma cells. We also confirmed that GRP-R is upstream of AKT2 and in turn, regulated N-myc expression via AKT2 in neuroblastoma cells. Functional assays demonstrated that attenuation of AKT2 impaired cell proliferation and anchorage-independent cell growth, and decreased the secretion of angiogenic factor VEGF in vitro. Furthermore, silencing AKT2 inhibited migration and invasion of neuroblastoma cells in vitro. Xenografts established by injecting AKT2 silenced human neuroblastoma cells into murine spleen expressed decreased levels of AKT2 and resulted in fewer liver metastases compared to controls in vivo. Hence, our study highlights the potential molecular mechanism(s) mediating the oncogenic role of GRP/GRP-R and demonstrates a novel role for AKT2 in neuroblastoma tumorigenesis, indicating that targeting the GRP/GRP-R/AKT2 axis may be important for developing novel therapeutics in the treatment of clinically aggressive neuroblastoma.

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Background The phosphatidylinositol 3-kinase (PI3K), a critical intracellular pathway, is negatively regulated by phosphatase and tensin homologue (PTEN). Integrin-linked kinase (ILK) induces phosphorylation of Akt leading to an increase in cell survival. However, a potential interaction between ILK and PTEN activity in neuroblastoma cells is unknown. We sought to examine the relationship between ILK and PTEN in the PI3K/Akt signaling pathway in neuroblastoma tumorigenesis. Methods The human neuroblastoma cell line, BE(2)-C, was transfected with small interfering (si) or short hairpin (sh) RNA to silence ILK expression. A plasmid containing the ILK wild type (ILK wt) gene was transfected to over express ILK. Cell proliferation was assessed, and anchorage independence was measured by soft agar assay. Insulin-like growth factor (IGF)-1 was used to stimulate the PI3K/Akt pathway. Protein levels were determined by Western blotting. Results Transient silencing of ILK produced correlative decreases in PTEN expression, cell proliferation and soft agar colony formation. Conversely, stably-transfected ILK knockdown cells showed an increase in phospho-Akt levels, leading to cell proliferation. Conclusions ILK plays an important role in the regulation of PI3K/Akt pathway via PTEN or an upstream effector of PTEN. The effects of ILK silencing on PTEN expression appear to be critically dependent on duration of ILK dysregulation.