The macrolide antibiotic rapamycin is a potentially novel anticancer agent that specifically inhibits the activity of a protein kinase (mTOR) that controls translation of proteins involved in cell cycle progression and survival. The response to mTOR inhibition in normal and malignant cells is qualitatively different: rapamycin causes G1 phase arrest and cytostasis in normal cells, whereas treatment of rhabdomyosarcoma (RMS) cells causes G1 arrest, and induces apoptosis. Results from the applicant's laboratory indicate that apoptosis occurs only in cells in which RB is functional, but in which p53 tumor suppressor function is abrogated. This differential confers a basis for tumor-selectivity for rapamycin. The major focus of the proposed studies is to understand how inhibition of mTOR signaling by rapamycin induces apoptosis, and how p53 protects cells. Using a series of human RMS cell lines with characterized p53 alleles, clones constructed with inducible p53, and murine embryo fibroblasts disrupted at p53 and/or RB loci, the applicant will test the following hypotheses: 1) Rapamycin induces apoptosis only in cells with mutated or abrogated p53 function. 2) p53 activates a G1 cell cycle checkpoint preventing cell death. 3) In cells with an abrogated p53 function, inhibition of mTOR leads to a decrease in survival factors or an increase in pro-apoptotic factors. By initiating a G1 checkpoint, p53 will modulate survival factors, thus preventing apoptosis. 4) Although mTOR activates translation of specific subsets of mRNA under two separate pathways, by directly phosphorylating PHAS-I and by indirectly activating ribosomal p70S6 kinase, the hypothesis is that rapamycin-induced growth arrest and apoptosis is a consequence of inhibition of the PHAS-I pathway. 5) IGF-I protection of RMS cells from rapamycin occurs through activating NF-kB by a pathway independent of mTOR, and is dependent on the level of expression of the type-I IGF receptor. The proposed studies will elucidate the role of mTOR signaling in cell survival, and why loss of p53 sensitizes cells to apoptosis. These studies will give insights into approaches that combine signal transduction inhibitors to give tumor-selective apoptosis based on loss of p53 function. The long-term goal of these studies is to develop alternative curative therapy for children with cancer that will avoid the often devastating events, associated with contemporary intensive chemo-radiotherapy regimens.
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