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We recently identified activation of phosphatidylinositol 3'-kinase (PI3K)/Akt as a novel predictor of poor outcome in neuroblastoma. Here, we investigated the effect of small-molecule PI3K inhibitors on chemosensitivity. We provide first evidence that PI3K inhibitors, for example PI103, synergize with various chemotherapeutics (Doxorubicin, Etoposide, Topotecan, Cisplatin, Vincristine and Taxol) to trigger apoptosis in neuroblastoma cells (combination index: high synergy). Mechanistic studies reveal that PI103 cooperates with Doxorubicin to reduce Mcl-1 expression and Bim(EL) phosphorylation and to upregulate Noxa and Bim(EL) levels. This shifted ratio of pro- and antiapoptotic Bcl-2 proteins results in increased Bax/Bak conformational change, loss of mitochondrial membrane potential, cytochrome c release, caspase activation and caspase-dependent apoptosis. Although Mcl-1 knockdown enhances Doxorubicin- and PI103-induced apoptosis, silencing of Noxa, Bax/Bak or p53 reduces apoptosis, underscoring the functional relevance of the Doxorubicin- and PI103-mediated modulation of these proteins for chemosensitization. Bcl-2 overexpression inhibits Bax activation, mitochondrial perturbations, cleavage of caspases and Bid, and apoptosis, confirming the central role of the mitochondrial pathway for chemosensitization. Interestingly, the broad-range caspase inhibitor zVAD.fmk does not interfere with Bax activation or mitochondrial outer membrane permeabilization, whereas it blocks caspase activation and apoptosis, thus placing mitochondrial events upstream of caspase activation. Importantly, PI103 and Doxorubicin cooperate to induce apoptosis and to suppress tumor growth in patients' derived primary neuroblastoma cells and in an in vivo neuroblastoma model, underlining the clinical relevance of the results. Thus, targeting PI3K presents a novel and promising strategy to sensitize neuroblastoma cells for chemotherapy-induced apoptosis, which has important implications for the development of targeted therapies for neuroblastoma.

Rhabdomyosarcoma (RMS) is a highly malignant tumor that is histologically related to skeletal muscle, yet genetic and molecular lesions underlying its genesis and progression remain largely unknown. In this study we have compared the molecular profiles of two different mouse models of RMS, each associated with a defined primary genetic defect known to play a role in rhabdomyosarcomagenesis in man. We report that RMS of heterozygous Patched1 (Ptch1) mice show less aggressive growth and a greater degree of differentiation than RMS of heterozygous p53 mice. By means of cDNA microarray analysis we demonstrate that RMS in Ptch1 mutants predominantly express a number of myogenic markers, including myogenic differentiation 1, myosin heavy chain, actin, troponin and tropomyosin, as well as genes associated with Hedgehog/Patched signaling like insulin-like growth factor 2, forkhead box gene Foxf1 and the growth arrest and DNA-damage-inducible gene Gadd45a. In sharp contrast, RMS in p53 mutants display higher expression levels of cell cycle-associated genes like cyclin B1, cyclin-dependent kinase 4 and the proliferation marker Ki-67. These results demonstrate that different causative mutations lead to distinct gene expression profiles in RMS, which appear to reflect their different biological characteristics. Our results provide a first step towards a molecular classification of different forms of RMS. If the described differences can be confirmed in human RMS our results will contribute to a new molecular taxonomy of this cancer, which will be critical for gene mutation- and expression-specific therapy.