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Purpose: The phosphoinositide 3-kinase (PI3K)/Akt pathway is frequently activated in human cancer and plays a crucial role in neuroblastoma biology. We were interested in gaining further insight into the potential of targeting PI3K/Akt signaling as a novel antiproliferative approach in neuroblastoma. Experimental Design: The expression pattern and functions of class I A PI3K isoforms were investigated in tumor samples and cell lines. Effects on cell survival and downstream signaling were analyzed following down-regulation of p110a or p110y in SH-SY5Y and LA-N-1 cells by means of RNA interference. Results: Overexpression of the catalytic p110y and regulatory p85a isoforms was detected in a panel of primary neuroblastoma samples and cell lines, compared with normal adrenal gland tissue. Although down-regulation of either p110a or p110y led to impaired cell growth, reduced expression of p110y also had a selective effect on the survival of SH-SY5Ycells. Decreased levels of p110y were found to induce apoptosis and lead to lower expression levels of antiapoptotic Bcl-2 family proteins. SH-SY5Y cells with decreased p110y levels also displayed reduced activation of ribosomal protein S6 kinase in response to stimulation with epidermal growth factor and insulin-like growth factor-I. Conclusions: Together, our data reveal a novel function of p110y in neuroblastoma growth and survival.

Targeting receptor tyrosine kinase signaling in acute myeloid leukemia The main goal of this work was therefore aimed at gaining deeper insight into RTK signaling in a panel of AML cell lines and patient blast cells. A broad potein expression analysis was set up in order to identify cancer-specific expression patterns of signaling molecules and to uncover potential molecular targets. Using different approaches the molecular function and the feasibility of targeting the candidate proteins was investigated. Novel specific inhibitors were tested and neutralizing antibodies and RNA interference (RNAi) were used to block or down-regulate the individual proteins. Our study of the IGF-IR/PI3K signaling system in AML cells uncovered a novel role for autocrine IGF-I signaling in the growth and survival of these cancer cells. Besides, we could show that targeting this signaling branch in combination with commonly used chemotherapeutical agents represents an interesting novel approach for AML therapy.As protein expression analysis revealed highly variable expression levels of the mammalian target of rapamycin (mTOR) in the panel of cells analyzed, further interest was laid on the role of mTOR in AML. Cells expressing low levels of mTOR were compared to cells expressing high levels of this protein and a siRNA screen was aimed at uncovering human kinases that modulate the sensitivity to the mTOR inhibitor rapamycin. Preliminary results suggest that mTOR plays a crucial role in cell growth and survival in a subset of AML cells and that rapamycin in combination with certain RTK or Syk/ZAP70 inhibitors has potential for AML therapy. In summary, our study of the RTK/PI3K signaling system in distinct human cancers has provided novel insights into the importance and the complexity of this network for tumor growth and survival. Moreover, the analysis of specific components of this crucial signaling network has uncovered potential molecular targets for future cancer therapy.

The control of cellular growth is tightly linked to the regulation of protein synthesis. A key function in translation initiation is fulfilled by the 5' cap binding eukaryotic initiation factor 4E (eIF4E), and dysregulation of eIF4E is associated with malignant transformation and tumorigenesis . In mammals, the activity of eIF4E is modulated by phosphorylation at Ser209 by mitogen-activated protein kinases (MAPK)-interacting kinases 1 and 2 (Mnk1 and Mnk2) , which themselves are activated by ERK and p38 MAPK in response to mitogens, cytokines or cellular stress . Whether phosphorylation of eIF4E at Ser209 exerts a positive or inhibitory effect on translation efficiency has remained controversial. Here we provide a genetic characterization of the Drosophila homolog of Mnk1/2, Lk6. Lk6 function is dispensable under a high protein diet, consistent with the recent finding that mice lacking both Mnk1 and Mnk2 are not growth-impaired . Interestingly, loss of Lk6 function causes a significant growth reduction when the amino acid content in the diet is reduced. Overexpression of Lk6 also results in growth inhibition in an eIF4E-dependent manner. We propose a model of eIF4E regulation that may reconcile the contradictory findings with regard to the role of phosphorylation by Mnk1/2.

AT/RTs (atypical teratoid/rhabdoid tumours) of the CNS (central nervous system) are childhood malignancies associated with poor survival rates due to resistance to conventional treatments such as chemotherapy. We characterized a panel of human AT/RT and MRT (malignant rhabdoid tumour) cell lines for expression of RTKs (receptor tyrosine kinases) and their involvement in tumour growth and survival. When compared with normal brain tissue, AT/RT cell lines overexpressed the IR (insulin receptor) and the IGFIR (insulin-like growth factor-I receptor). Moreover, insulin was secreted by AT/RT cells grown in serum-free medium. Insulin potently activated Akt (also called protein kinase B) in AT/RT cells, as compared with other growth factors, such as epidermal growth factor. Pharmacological inhibitors, neutralizing antibodies, or RNAi (RNA interference) targeting the IR impaired the growth of AT/RT cell lines and induced apoptosis. Inhibitors of the PI3K (phosphoinositide 3-kinase)/Akt pathway also impaired basal and insulin-stimulated AT/RT cell proliferation. Experiments using RNAi and isoform-specific pharmacological inhibitors established a key role for the class I(A) PI3K p110alpha isoform in AT/RT cell growth and insulin signalling. Taken together, our results reveal a novel role for autocrine signalling by insulin and the IR in growth and survival of malignant human CNS tumour cells via the PI3K/Akt pathway.