Effects of small molecule inhibitors of PI3K/Akt/mTOR signaling on neuroblastoma growth in vitro and in vivo

Abstract

Activation of the PI3K/Akt signaling pathway is correlated with poor prognosis in neuroblastoma, the most common and deadly extracranial tumor of childhood. In this study, we show that the small-molecule inhibitors of phosphoinositidedependent protein kinase-1 (PDK1) OSU03012 and the dual class I PI3K/mTOR inhibitor PI103 have profound effects on neuroblastoma survival in vitro and in vivo. Both OSU03012 and PI103 inhibited neuroblastoma growth in vitro. In treated cells, OSU03012 induced apoptosis and an S phase cell cycle arrest, whereas only minor apoptosis was detected in PI103 treated cells together with a G1 arrest. Both OSU03012 and PI103 downregulated phosphorylation of Akt and inhibited the downstream targets glycogen synthase kinase-3b (GSK3b) and p70 S6 kinase-1 (S6K1), as well as downregulated the expression of cyclin D1 and Mycn protein. Neuroblastoma cells expressing high levels of Mycn were more sensitive to OSU03012 or PI103 compared with cells expressing low Mycn levels. Both compounds significantly inhibited the growth of established, subcutaneous MYCN-amplified neuroblastoma xenografts in nude NMRI nu/nu mice. These results suggest that inhibition of the PI3K/Akt signaling pathway represent a clinical relevant target for the treatment of patients with high-risk MYCN-amplified neuroblastoma.Neuroblastomas are peripheral nervous system tumors that originate in the neural crest and are most commonly found in the adrenal medulla or along the sympathetic chain. 1 These tumors show heterogeneous biological and clinical features, in which a subset is prone to regress through spontaneous apoptosis with little or no therapy while another subset differentiates over time to benign ganglioneuromas. However, the majority of neuroblastomas are malignant with metastatic spread that is difficult to cure with current treatment modalities.2 Amplification of the MYCN gene, which occurs in 40-50% of the high-risk neuroblastoma cases, remains the major key predictor of poor outcome and is associated with advanced-stage disease, rapid tumor progression and low survival.3 Advanced-stage tumors and those with MYCN gene amplification typically show emergence of treatment resistance and new treatment alternatives for these patients are highly warranted.Abnormal activation of the PI3K/Akt signaling pathway is a common event in human cancers and has been validated through epidemiological and experimental studies as being essential for several human tumors, including neuroblastoma.4-6 PI3K is a family of lipid kinases, heterodimeric proteins composed of one catalytic and one regulatory subunit. In the class IA PI3Ks, there are currently three isoforms of the catalytic (p110a, b, and d) and five of the regulatory (p50a, p55a, p85a, p85b, and p55c) subunits, where p50a and p55a are splice variants of p85a. PI3Ks functions as signal transducers downstream of cell-surface receptors, such as receptor tyrosine kinases. Activated PI3Ks phosphorylate the lipid phosphatidyl-inositol 4,5-biphosphate (PIP2) to generate phosphatidyl-...

Functional MYCN signature predicts outcome of neuroblastoma irrespective of MYCN amplification

“…Destabilization of MYCN protein levels by inactivation of PI3K has been observed in neuroblastoma cell lines (8,9). However, to our knowledge this has not been investigated in human neuroblastoma series.…”

“…Accordingly, activation of the PI3K/AKT pathway in neuroblastoma cell lines resulted in stabilization of MYCN protein (7,8). Inactivation of the pathway using a PI3K inhibitor resulted in reduced levels of MYCN (9). At another level, FBXW7 (F-box/WD repeat-containing protein 7) and AURKA (aurora kinase A) are involved in MYCN protein stability (10).…”

The N-myc Oncogene: Maximizing its Targets, Regulation, and Therapeutic Potential

“…This primarily occurs during the mitotic phase of the cell cycle and after phosphorylation of N-myc (S62 and T58) by the cell-cycle kinase cyclin B/CDK1 (70) and by glycogen synthase kinase3b (GSK3b). Because GSK3b is inactivated by AKT, activation of the PI3K/AKT pathway results in stabilization of the N-myc protein (71,72), and inactivation of the pathway using a PI3K inhibitor has been shown to reduce levels of N-myc (73). This is one therapeutically targetable pathway that may be relevant in other N-myc-driven and PI3K/AKT pathway implicated tumors as well.…”

Cytostatic in vitro Effects of DTCM-Glutarimide on Bladder Carcinoma Cells

“…On the other hand, flow cytometry analysis demonstrated sustained G1 arrest in 5637 and T24 cells after treatment with DTCM-g. Similarly, the dual PI3 kinase/mTOR inhibitor PI-103 induced a G1 arrest but no apoptosis in glioma (Fan et al, 2006) and G1 arrest with only minor apoptosis in neuroblastoma cells treated with the same inhibitor (Segerstrom et al, 2011).…”

“…Also, the inhibition of PDK1 by phenothiazines resulted in suppression of EGF-induced cell growth in ovary cancer cells and inhibited growth in other several cancer cell lines, including melanoma (SK-MEL-28), colon (HT29, Colo205, SW480, HCT116) and breast cancer (MCF7) (Choi et al, 2008). Likewise, the inhibitor OSU03012 had profound effects on medulloblastoma (Baryawno et al, 2010) and neuroblastoma survival in vitro and in vivo (Segerstrom et al, 2011). The PDK1 inhibitors, BX-795, BX-912, and BX-320 have also shown to inhibit growth in a wide panel of cell lines (Feldman et al, 2005).…”