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.

Regulated cell proliferation is a crucial prerequisite for Schwann cells to achieve myelination in development and regeneration. In the present study, we have investigated the function of the cell cycle inhibitors p21 and p16 as potential regulators of Schwann cell proliferation, using p21- or p16-deficient mice. We report that both inhibitors are required for proper withdrawal of Schwann cells from the cell cycle during development and following injury. Postnatal Schwann cells express p21 exclusively in the cytoplasm, first detectable at postnatal day 7. This cytoplasmic p21 expression is necessary for proper Schwann cell proliferation control in the late development of peripheral nerves. After axonal damage, p21 is found in Schwann cell nuclei during the initiation of the proliferation period. This stage is critically regulated by p21, since loss of p21 leads to a strong increase in Schwann cell proliferation. Unexpectedly, p21 levels are upregulated in this phase suggesting that the role of p21 may be more complex than purely inhibitory for the Schwann cell cycle. However, inhibition of Schwann cell proliferation is the overriding crucial function of p21 and p16 in peripheral nerves as revealed by the consequences of loss-of-function in development and after injury. Different mechanisms appear to underlie the inhibitory function, depending on whether p21 is cytoplasmic or nuclear.

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.

The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancer and plays a crucial role in glioblastoma biology. We were interested in gaining further insight into the potential of targeting PI3K isoforms as a novel anti-tumor approach in glioblastoma. Consistent expression of the PI3K catalytic isoform PI3K p110α was detected in a panel of glioblastoma patient samples. In contrast, PI3K p110β expression was only rarely detected in glioblastoma patient samples. The expression of a module comprising the epidermal growth factor receptor (EGFR)/PI3K p110α/phosphorylated ribosomal S6 protein (p-S6) was correlated with shorter patient survival. Inhibition of PI3K p110α activity impaired the anchorage-dependent growth of glioblastoma cells and induced tumor regression in vivo. Inhibition of PI3K p110α or PI3K p110β also led to impaired anchorage-independent growth, a decreased migratory capacity of glioblastoma cells, and reduced the activation of the Akt/mTOR pathway. These effects were selective, because targeting of PI3K p110δ did not result in a comparable impairment of glioblastoma tumorigenic properties. Together, our data reveal that drugs targeting PI3K p110α can reduce growth in a subset of glioblastoma tumors characterized by the expression of EGFR/PI3K p110α/p-S6.

The potential of the novel insulin‐like growth factor receptor (IGF‐IR) inhibitor NVP‐AEW541 as an antiproliferative agent in human neuroblastoma was investigated. Proliferation of a panel of neuroblastoma cell lines was inhibited by NVP‐AEW541 with IC50 values ranging from 0.15 to 5 μM. Experiments using an IGF‐IR neutralizing antibody confirmed that the IGF‐IR was essential to support growth of neuroblastoma cell lines. The expression levels of the IGF‐IR in individual neuroblastoma cell lines did not correlate with the sensitivities to NVP‐AEW541, while coexpression of the IGF‐IR and the insulin receptor (IR) correlated with lower sensitivity to the inhibitor in some cell lines. Intriguingly, high levels of activation of Akt/protein kinase B (PKB) and phosphorylation of the ribosomal S6 protein were observed in neuroblastoma cell lines with decreased sensitivities to NVP‐AEW541. Inhibition of Akt/PKB activity restored the sensitivity of neuroblastoma cells to the IGF‐IR inhibitor. Transfection of neuroblastoma cells with activated Akt or ribosomal protein S6 kinase (S6K) decreased the sensitivity of the cells to NVP‐AEW541. IGF‐I‐stimulated proliferation of neuroblastoma cell lines was completely blocked by NVP‐AEW541, or by a combination of an inhibitor of phosphoinositide 3‐kinase and rapamycin. In addition to its antiproliferative effects, NVP‐AEW541 sensitized neuroblastoma cells to cisplatin‐induced apoptosis. Together, our data demonstrate that NVP‐AEW541 in combination with Akt/PKB inhibitors or chemotherapeutic agents may represent a novel approach to target human neuroblastoma cell proliferation. © 2006 Wiley‐Liss, Inc.

As insulin-like growth factor (IGF) signaling has been recognized to play an important role in human cancer, the IGF-I receptor (IGF-IR) is currently the focus of intensive research aimed at developing novel antitumor agents. The IGF system is frequently deregulated in cancer cells by the establishment of autocrine loops involving IGF-I or -II and/or IGF-IR over-expression. Moreover, epidemiological studies have suggested a link between elevated IGF levels and the development of major human malignancies, such as breast, colon, lung and prostate cancer. Experimental therapies aimed at inhibiting IGF signaling in human tumors involve various approaches, including neutralizing antibodies and pharmacological inhibitors of IGF-IR kinase activity. Although there are numerous reports describing the antitumor activity of such agents against human cancer cell lines propagated in vitro or in experimental animals, it remains unclear how soon the existing drugs will have a demonstrable effect in patients. In this review, we will discuss the evidence implicating the IGF signaling system in the pathology of human cancer and the various strategies that have so far been developed to target the IGF-IR.

Background: Eight human catalytic phosphoinositide 3-kinase (PI3K) isoforms exist which are subdivided into three classes. While class I isoforms have been wellstudied in cancer, little is known about the functions of class II PI3Ks. Materials and Methods: The expression pattern and functions of the class II PI3KC2β isoform were investigated in a panel of tumour samples and cell lines. Results: Overexpression of PI3KC2β was found in subsets of tumours and cell lines from acute myeloid leukemia (AML), glioblastoma multiforme (GBM), medulloblastoma (MB), neuroblastoma (NB), and small cell lung cancer (SCLC). Specific pharmacological inhibitors of PI3KC2β or RNA interference impaired proliferation of a panel of human cancer cell lines and primary cultures. Inhibition of PI3KC2β also induced apoptosis and sensitised the cancer cells to chemotherapeutic agents. Conclusion: Together, these datashow that PI3KC2β contributes to proliferation and survival in AML, brain tumours and neuroendocrine tumours, and may represent a novel target in these malignancies.Phosphoinositide 3-kinases (PI3Ks) play an essential role in the signal transduction events initiated by the binding of extracellular signals to their cell surface receptors (1-3). Cellular responses controlled by PI3Ks are extremely diverse, including mitogenesis and proliferation, protection from apoptosis and cell motility (1-3). There are eight known PI3Ks in humans, which have been subdivided into three classes, based on structural homology and in vitro substrate specificity (4-7). Class I A comprises three highly homologous isoforms, p110α (8), p110β (9) and p110δ (10, 11), which exist as a heterodimeric complex with a regulatory subunit containing two SRC homology-2 (SH2) domains, mediating enzyme association with phosphotyrosine residues in the cytoplasmic domains of activated polypeptide growth factor receptors (12)(13)(14). All class I PI3Ks function as PtdIns(4,5)P 2 3-kinase in vivo, upon activation by receptor tyrosine kinases or serpentine receptors (15, 16). PtdIns(3,4,5)P 3 serves as a docking site for the serine/threonine protein kinase phosphoinositide-dependent protein kinase-1 (PDK-1), which is activated upon binding (17). Several protein kinases have been identified as 3217