neuroblastoma ͉ synthetic lethal T wo genes are considered to be "synthetically lethal" if mutation of either gene alone is compatible with viability but simultaneous mutation of both genes causes death. This mechanism is best described for loss of function genes but also exits for gain of function genes. For example gene A can become essential for survival if gene B is over-expressed. This mechanism is potentially very attractive for the development of targeted anti cancer compounds that could specifically kill tumor cells while leaving normal cells alive (1-3). However, examples of synthetic lethal oncogenes in human tumors are hardly documented.Neuroblastomas are embryonal tumors that originate from the developing sympathetic nervous system, and although neuroblastomas have a low incidence, they are the second cause of cancer related deaths in children (4, 5). The MYCN gene is amplified in 20-30% of neuroblastoma tumors and amplification strongly correlates with a bad prognosis. MYC genes are potent oncogenes that drive unrestrained cell growth and proliferation. They function as transcription factors that cause up-regulation or repression of genes involved in a variety of oncogenetic pathways. Recently MYC genes were also shown to control protein expression through mRNA translation and to directly regulate DNA replication (6-9). Apart from the function in oncogenesis, MYC genes have also been described to induce apoptosis if over-expressed in non MYC-amplified cells (10). This could indicate that the amplification of the MYC oncogenes requires a specific genetic background and that there could be synthetic lethal relations with other (onco)genes.Cell cycle aberrations occur in all tumors and many targeted compounds inhibiting specific cell cycle kinases have been developed (11,12). These efforts were based on the idea that the targeting of aberrant cell cycle checkpoints in cancer cells could lead to tumor growth inhibition and cell death (13). Several studies have recently shown that most cell cycle kinases are not essential for cell survival in vitro and in vivo (14). Cyclindependent kinase 2 (CDK2) was thought to be a crucial regulator of S-phase progression and was therefore evaluated as an anticancer drug target. Tetsu et al. however showed that CDK2 inhibition in several cancer cell types did not result in cell death, and Santamaria et al. showed that genetic ablation of CDK2 in mice could be compensated for by CDK1 (15,16). These findings severely reduced optimism about CDK2 as therapeutic target.Several cell cycle aberrations involving G1-regulating genes have been identified in neuroblastomas. Cyclin D1 and CDK4 gene amplifications occur at a low frequency, and a CDK6 mutation that inactivates p16-binding has been found (17)(18)(19). Cyclin D1 was found to be extremely over-expressed in about 75% of neuroblastoma tumors. Inhibition of the G1 regulating genes CDK4 or cyclin D1 in neuroblastoma cell lines resulted in restoration of the G1 checkpoint and subsequent neuronal differentiation (20). The ab...
Doublecortin-like kinase-long (DCLK-long) and doublecortin-like (DCL) are two splice variants of DCLK gene. DCL and DCLK-long are microtubule-associated proteins with specific expression in proliferative neural progenitor cells. We have tested the hypothesis that knockdown of DCL/DCLKlong by RNA interference technology will induce cell death in neuroblastoma (NB) cells. First, we analyzed the expression of DCL and DCLK-long in several human neuroblastic tumors, other tumors, and normal tissues, revealing high expression of both DCL and DCLK-long in NB and glioma. Secondly, gene expression profiling revealed numerous differentially expressed genes indicating apoptosis induction after DCL/DCLK-long knockdown in NB cells. Finally, apoptosis was confirmed by time-lapse imaging of phosphatidylserine translocation, caspase-3 activation, live/dead double staining assays, and fluorescence-activated cell sorting. Together, our results suggest that silencing DCL/DCLK-long induces apoptosis in NB cells.
The BIRC5 (Survivin) gene is located at chromosome 17q in the region that is frequently gained in high risk neuroblastoma. BIRC5 is strongly over expressed in neuroblastoma tumour samples, which correlates to a poor prognosis. We recently validated BIRC5 as a potential therapeutic target by showing that targeted knock down with shRNA's triggers an apoptotic response through mitotic catastrophe. We now tested YM155, a novel small molecule selective BIRC5 suppressant that is currently in phase I/II clinical trials. Drug response curves showed IC50 values in the low nM range (median: 35 nM, range: 0.5-> 10,000 nM) in a panel of 23 neuroblastoma cell lines and four TIC-lines, which resulted from an apoptotic response. Nine out of 23 cell lines were relatively resistant to YM155 with IC50 values > 200 nM, although in the same cells shRNA mediated knock down of BIRC5 caused massive apoptosis. Analysis of differentially expressed genes between five most sensitive and five most resistant cell lines using Affymetrix mRNA expression data revealed ABCB1 (MDR1) as the most predictive gene for resistance to YM155. Inhibition of the multi-drug resistance pump ABCB1 with cyclosporine or knockdown with shRNA prior to treatment with YM155 demonstrated that cell lines with ABCB1 expression became 27-695 times more sensitive to YM155 treatment. We conclude that most neuroblastoma cell lines are sensitive to YM155 in the low nM range and that resistant cells can be sensitised by ABCB1 inhibitors. Therefore YM155 is a promising novel compound for treatment of neuroblastoma with low ABCB1 expression.
BIRC5 (survivin) is one of the genes located on chromosome arm 17q in the region that is often gained in neuroblastoma. BIRC5 is a protein in the intrinsic apoptotic pathway that interacts with XIAP and DIABLO leading to caspase-3 and caspase-9 inactivation. BIRC5 is also involved in stabilizing the microtubule-kinetochore dynamics. Based on the Affymetrix mRNA expression data, we here show that BIRC5 expression is strongly upregulated in neuroblastoma compared with normal tissues, adult malignancies, and non-malignant fetal adrenal neuroblasts. The overexpression of BIRC5 correlates with an unfavorable prognosis independent of the presence of 17q gain. Silencing of BIRC5 in neuroblastoma cell lines by various antisense molecules resulted in massive apoptosis as measured by PARP cleavage and FACS analysis. As both the intrinsic apoptotic pathway and the chromosomal passenger complex can be therapeutically targeted, we investigated in which of them BIRC5 exerted its essential anti-apoptotic role. Immunofluorescence analysis of neuroblastoma cells after BIRC5 silencing showed formation of multinucleated cells indicating mitotic catastrophe, which leads to apoptosis via P53 and CASP2. We show that BIRC5 silencing indeed resulted in activation of P53 and we could rescue apoptosis by CASP2 inhibition. We conclude that BIRC5 stabilizes the microtubules in the chromosomal passenger complex in neuroblastoma and that the apoptotic response results from mitotic catastrophe, which makes BIRC5 an interesting target for therapy.
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