Neuroblastoma (NB) is the third most common malignancy in childhood and accounts for about 15% of cancer-related deaths in children. One of the most striking abnormalities found in NB tumors is the amplification of the MYCN oncogene, which strongly correlates with more aggressive tumors and poor prognosis of late stage disease. The complete absence of effective treatments for high-risk (late stage) NB patients indicates the need for novel therapeutic approaches, including drugs that block MYCN expression. The primary objective of our previous work has been to evaluate the polyamine biosynthetic pathway as an alternative target for NB therapy. Central to our investigation was the evaluation of two clinically tested polyamine inhibitors, DFMO and SAM486A, which specifically inhibit the polyamine biosynthetic enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), respectively. Since MYCN has been shown to activate ODC gene expression, we hypothesized that ODC and polyamines might play a role in MYCN-amplified NB tumorigenesis. Our initial results showed that the depletion of polyamines in MYCN-amplified NB cells caused G1 cell cycle arrest and affected p27Kip1 levels, retinoblastoma protein Rb phosphorylation, and most intriguingly, blocked MYCN expression, thus providing a rationale for the potential use of polyamine inhibitors in late-stage NB patients. In the current proposal we outline studies that examine how MYCN, ODC, and polyamines regulate important cell cycle and cell survival pathways in NB cells.
In Specific Aim 1 we will study whether MYCN overexpression in NB cells impacts endogenous ODC and other metabolic enzymes of polyamine metabolism, and whether this translates into changes in polyamine pools, cell cycle progression, and cell proliferation. These studies will be performed using three unique NB cell lines, in which MYCN expression can be switched on/off by adding tetracycline.
In Specific Aim 2 we will examine the role of polyamines in cell cycle regulation and PI3K/Akt cell survival activation in NB cells. Specifically, we will determine the differential effects of individual polyamines (putrescine, spermidine, and spermine) on the regulation and phosphorylation/activation of key signaling proteins including MYCN, p27Kip1, Cdk2, Akt, and mTOR.
In Specific Aim 3 we will elucidate the role of ODC in cell signaling by examining its interaction with two new cellular proteins. The physiological relevance of these interactions will be verified by mutational analyses and a series of cell-based biological assays. The proposed methods and techniques to pursue this work are: Northern blot, RT-PCR, nuclear run-ons, messenger RNA half-life studies, Western blot, yeast two-hybrid, co-immunoprecipitation, GST-pull down, site-directed mutagenesis, DNA cloning, confocal laser microscopy, enzyme and in vitro kinase assays, flow cytometry, and HPLC. At the conclusion of this research, we will have uncovered how ODC and individual polyamines regulate important molecular events that control cell cycle and cell survival pathways of NB cells. Our research will be useful in the development of novel treatments for the most aggressive forms of the childhood cancer neuroblastoma. ? ? ?
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