The long-term goal of this project is to develop novel therapeutic approaches for the treatment of neuroblastoma, the most common extra-cranial tumor of early childhood. Ceramides have been shown to be involved in the differentiation of neuroblastoma cell lines in vitro [1, 2], and we will therefore focus specifically on the mechanisms by which ceramide mediates differentiation and telomerase inhibition in human neuroblastoma cells. Retinoids such as alltrans-retinoic acid (ATRA) and 13-cis-retinoic acid have been shown to induce differentiation, growth arrest, and apoptosis in neuroblastoma cell lines in vitro and in-vivo [3-5]. Retinoids exert their biologic effects through binding to retinoic acid receptors (RAR) and retinoid X receptors (RXR) thereby modulating gene expression [6, 7]. Multiple cellular mechanisms are activated, including the generation of the sphingolipid second messenger ceramide. Ceramide mediates important cellular activities such as induction of cell differentiation [8-10], growth arrest [11], senescence [12, 13], and apoptosis [14-116]. It has been shown to regulate the activity of various biochemical and molecular targets, including serine/threonine phosphatases of the protein phosphatase 2A (PP2A) and PP1 families designated as ceramide activated protein phosphatases (CAPPs) [17-20]. Recent studies from our group have demonstrated the ability of ceramide to inhibit telomerase in lung adenocarcinoma cells [21, 22]. Telomerase is a ribonucleoprotein enzyme complex whose function is to add hexameric nucleotide repeats (TTAGGG)n to the ends of chromosomes [23, 24]. Telomeric loss is associated with senescence and cell death [25-27]. Most immortalized cells and malignant cells have increased telomerase activity [28-31]. While telomerase itself is not sufficient to cause neoplasia, its presence enables malignant cells to maintain telomere length, allowing infinite replicative capacity [32]. Increased telomerase activity is emerging as a prognostic factor in neuroblastoma, associated with advanced stage, MYCN amplification and poor outcome [33-37]. Telomerase has been proposed as a key novel target for anti-cancer chemotherapy developmen1. It is, thus, reasonable to hypothesize that retinoic acid's effects on cell growth, differentiation, and apoptosis are mediated by ceramide and that modulating cellular ceramide levels will have profound therapeutic implications. The long-term goal of this research will be to regulate the ceramide pathway and inhibit telomerase in neuroblastoma by using translational studies. These studies should significantly enhance the treatment of this devastating childhood disease.
The specific aims of this proposal are:
Specific aim 1. To determine if the generation of endogenous ceramide is necessary and/or sufficient to induce differentiation.
This specific aim will test the hypothesis that ceramide alone is sufficient to induce differentiation. The role of endogenous ceramide on differentiation will be studied. This will be performed by: a) Measuring the levels of endogenous ceramide in response to retinoids, b) Determining if blocking ceramide synthesis with the inhibitors fumonisin and myriocin will inhibit retinoid-induced differentiation. We will observe for the morphological changes of differentiation, and measure markers of neuronal differentiation including expression of RET, and ENO. c) Determining if endogenous ceramide is sufficient to induce differentiation will be accomplished by transfecting cells with bacterial sphingomyelinase (b-SMase) to elevate ceramide levels. These studies will serve to establish the critical role of ceramide in neuroblastoma differentiation.
Specific aim 2. To determine the molecular mechanisms and signaling pathways by which ceramide mediates differentiation in human neuroblastoma cells:
This aim will test the hypothesis that the changes in gene expression and cell cycle that occur during retinoid induced differentiation are mediated by ceramide. Therefore, we will a) study the effects of ceramide treatment on the cell cycle and on the expression of genes recognized to be induced by retinoic acid including MYCN, retinoblastoma (RB), B-myb, neurotrophin receptors (trk-A, trk-B, trk-C), and the cyclin dependent kinase inhibitor, p27Kipl, b) To determine if ceramide s effects are due to activation of phosphatases, we will block CAPPs with protein phosphatase inhibitors and study the effects on differentiation, cell cycle arrest and gene expression. These studies will establish that the ceramide pathway is indeed integral for neuroblastoma differentiation.
Specific aim 3. To determine if retinoic acid mediated telomerase inhibition is due to ceramide, and determine the molecular mechanisms and signalling pathways involved: We have recently shown that ceramide inhibits telomerase activity in lung adenocarcinoma cells [21, 22]. Our preliminary experiments also demonstrate the inhibition of telomerase by retinoic acid in neuroblastoma. Therefore, we hypothesize that retinoic acid inhibits telomerase via ceramide.
This specific aim will: a) examine the dose response and time course of telomerase inhibition in these cells, b) To determine if endogenous ceramide generation is necessary for telomerase inhibition, its generation will be blocked by treatment with fumonisin and myriocin; and telomerase activity will be measured, c) In order to test the hypothesis that de-phosphorylation of hTERT (the catalytic subunit of telomerase) by ceramide inhibits telomerase activity, i) we will determine the effects of ceramide on the phosphorylation status of hTERT, ii) treat with phosphatase inhibitors, and iii) use phosphorylation site mutants of hTERT. These studies will give us important insights into the mechanisms of ceramide mediated telomerase inhibition in neuroblastoma and may allow the discovery of novel and groundbreaking therapeutic modalities for the treatment of this disease.
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