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Histone deacetylase (HDAC) inhibitors are effective in MYCN-driven cancers, because of a unique need for HDAC recruitment by the MYCN oncogenic signal. However, HDAC inhibitors are much more effective in combination with other anti-cancer agents. To identify novel compounds which act synergistically with HDAC inhibitor, such as suberanoyl hydroxamic acid (SAHA), we performed a cell-based, high-throughput drug screen of 10,560 small molecule compounds from a drug-like diversity library and identified a small molecule compound (SE486-11) which synergistically enhanced the cytotoxic effects of SAHA. Effects of drug combinations on cell viability, proliferation, apoptosis and colony forming were assessed in a panel of neuroblastoma cell lines. Treatment with SAHA and SE486-11 increased MYCN ubiquitination and degradation, and markedly inhibited tumorigenesis in neuroblastoma xenografts, and, MYCN transgenic zebrafish and mice. The combination reduced ubiquitin-specific protease 5 (USP5) levels and increased unanchored polyubiquitin chains. Overexpression of USP5 rescued neuroblastoma cells from the cytopathic effects of the combination and reduced unanchored polyubiquitin, suggesting USP5 is a therapeutic target of the combination. SAHA and SE486-11 directly bound to USP5 and the drug combination exhibited a 100-fold higher binding to USP5 than individual drugs alone in microscale thermophoresis assays. MYCN bound to the USP5 promoter and induced USP5 gene expression suggesting that USP5 and MYCN expression created a forward positive feedback loop in neuroblastoma cells. Thus, USP5 acts as an oncogenic cofactor with MYCN in neuroblastoma and the novel combination of HDAC inhibitor with SE486-11 represents a novel therapeutic approach for the treatment of MYCN-driven neuroblastoma.

The N-Myc oncoprotein induces neuroblastoma, which arises from undifferentiated neuroblasts in the sympathetic nervous system, by modulating gene and protein expression and consequently causing cell differentiation block and cell proliferation. The class IIa histone deacetylase 5 (HDAC5) represses gene transcription, and blocks myoblast, osteoblast and leukemia cell differentiation. Here we showed that N-Myc upregulated HDAC5 expression in neuroblastoma cells. Conversely, HDAC5 repressed the ubiquitin-protein ligase NEDD4 gene expression, increased Aurora A gene expression and consequently upregulated N-Myc protein expression. Genome-wide gene expression analysis and protein co-immunoprecipitation assays revealed that HDAC5 and N-Myc repressed the expression of a common subset of genes by forming a protein complex, whereas HDAC5 and the class III HDAC SIRT2 independently repressed the expression of another common subset of genes without forming a protein complex. Moreover, HDAC5 blocked differentiation and induced proliferation in neuroblastoma cells. Taken together, our data identify HDAC5 as a novel co-factor in N-Myc oncogenesis, and provide the evidence for the potential application of HDAC5 inhibitors in the therapy of N-Myc-induced neuroblastoma and potentially other c-Myc-induced malignancies.

Embryonal cancer can arise from postnatally persistent embryonal remnant or rest cells, which are uniquely characterized by the absence of p53 mutations. Perinatal overexpression of the MycN oncoprotein in embryonal cancer precursor cells causes postnatal rests, and later tumor formation through unknown mechanisms. However, overexpression of Myc in adult tissues normally activates apoptosis and/or senescence signals as an organismal defense mechanism against cancer. Here, we show that perinatal neuroblastoma precursor cells exhibited a transiently diminished p53 response to MycN oncoprotein stress and resistance to trophic factor withdrawal, compared with their adult counterpart cells from the TH-MYCN(+/+) transgenic mouse model of neuroblastoma. The adult stem cell maintenance factor and Polycomb group protein, Bmi1 (B-cell-specific Moloney murine leukemia virus integration site), had a critical role at neuroblastoma initiation in the model, by repressing p53 responses in precursor cells. We further show in neuroblastoma tumor cells that Bmi1 could directly bind p53 in a complex with other Polycomb complex proteins, Ring1A or Ring1B, leading to increased p53 ubiquitination and degradation. Repressed p53 signal responses were also seen in precursor cells for other embryonal cancer types, medulloblastoma and acute lymphoblastic leukemia. Collectively, these date indicate a general mechanism for p53 inactivation in some embryonal cell types and consequent susceptibility to MycN oncogenesis at the point of embryonal tumor initiation.

The family of tripartite-motif (TRIM) proteins are involved in diverse cellular processes, but are often characterized by critical protein-protein interactions necessary for their function. TRIM16 is induced in different cancer types, when the cancer cell is forced to proceed down a differentiation pathway. We have identified TRIM16 as a DNA-binding protein with histone acetylase activity, which is required for the retinoic acid receptor β(2) transcriptional response in retinoid-treated cancer cells. In this study, we show that overexpressed TRIM16 reduced neuroblastoma cell growth, enhanced retinoid-induced differentiation and reduced tumourigenicity in vivo. TRIM16 was only expressed in the differentiated ganglion cell component of primary human neuroblastoma tumour tissues. TRIM16 bound directly to cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells. TRIM16 reduced cell motility and this required downregulation of vimentin. Retinoid treatment and enforced overexpression caused TRIM16 to translocate to the nucleus, and bind to and downregulate nuclear E2F1, required for cell replication. This study, for the first time, demonstrates that TRIM16 acts as a tumour suppressor, affecting neuritic differentiation, cell migration and replication through interactions with cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells.

Medulloblastoma tumorigenesis caused by inactivating mutations in the PATCHED1 (PTCH1) gene is initiated by persistently activated Sonic Hedgehog (Shh) signaling in granule neuron precursors (GNPs) during the late stages of cerebellar development. Both normal cerebellar development and Shh-driven medulloblastoma tumorigenesis require N-Myc expression. However, the mechanisms by which N-Myc affects the stages of medulloblastoma initiation and progression are unknown. Here we used a mouse model of Ptch1 heterozygosity and medulloblastoma to show that increased N-Myc expression characterized the earliest selection of focal GNP hyperplasia destined for later tumor progression. Step-wise loss of Ptch1 expression, from tumor initiation to progression, led to incremental increases in N-Myc protein, rather than mRNA, expression. Increased N-Myc resulted in enhanced proliferation and death resistance of perinatal GNPs at tumor initiation. Sequential N-Myc protein phosphorylation at serine-62 and serine-62/threonine-58 characterized the early and late stages of medulloblastoma tumorigenesis, respectively. Shh pathway activation led to increased Myc protein stability and reduced expression of key regulatory factors. Taken together our data identify N-Myc protein stability as the result of loss of Ptch1, which distinguishes normal cerebellar development from medulloblastoma tumorigenesis.

In the childhood cancer neuroblastoma (NB), the level of expression of the multidrug resistance-associated protein (MRP1) gene is strongly correlated with expression of the MYCN oncogene in primary NB tumors, suggesting that MRP1 may be a target for MYCN-mediated gene regulation. In this study, we show that MYCN induction in human NB cells results in increased MRP1 mRNA and protein levels, which in turn is accompanied by increased drug resistance and enhanced MRP1-mediated drug efflux. Furthermore, luciferase activity from MRP1 promoter/luciferase gene reporter constructs was significantly increased in NB cells with exogenous overexpression of MYCN, whereas activity was decreased in NB cells stably transfected with MYCN-antisense vectors. Decreased luciferase activity was observed with promoter constructs that lacked one or two E-box sequences or had E-box double point mutations, while a truncated MRP1 promoter lacking all three E-boxes exhibited only basal levels of activity. Specific electrophoretic mobility shifts of MRP1 E-box sequences were detected with nuclear extracts from NB cells with MYCN overexpression, and complex formation was inhibited with the addition of antibodies directed against MYCN or MYC. These findings indicate that by interacting with E-box elements within the promoter, MYCN can upregulate MRP1 expression and modulate drug resistance in NB.

We have previously shown that ectopic overexpression of retinoic acid receptor (RAR) subtypes alpha, beta and gamma in human neuroblastoma cells had different effects on growth and retinoid sensitivity. Only overexpressed RAR beta induced profound growth inhibition in the absence of additional retinoid, and increased retinoid sensitivity. In this study, we measured mRNA expression levels of RAR alpha, beta, and gamma in 50 primary neuroblastoma tumor samples, and found a strong correlation between favorable patient prognosis and high-level RAR beta expression. Human neuroblastoma cells transfected with a vector expressing RAR beta demonstrated irreversible growth arrest following a 1 week exposure to all-transretinoic acid, whereas control cells continued to proliferate. In the absence of additional retinoid, RAR beta transfectants demonstrated a higher proportion of cells in the G0/G1 phase of the cell cycle, increased p21WAF1/CIP1 expression and specific binding to a retinoic acid response element. These were changes which we also observed in control neuroblastoma cells following retinoid treatment. Our data indicate that RAR beta is an important factor mediating the growth inhibitory effects of retinoids in neuroblastoma cells. The favorable effect of high-level RAR beta expression on prognosis in primary tumor tissue may occur through RAR beta effects on p21 expression and consequent G0/G1 cell cycle arrest.

We have recently shown a close correlation between expression of the Multidrug Resistance-associated Protein (MRP) gene and the MYCN oncogene and provided evidence that high MRP expression is a powerful independent predictor of poor outcome in neuroblastoma (Norris et al., New Engl. J. Med., 334, 231-238, 1996). The effect of MYCN down-regulation on MRP expression and response to cytotoxic drugs was investigated in NBL-S neuroblastoma cells transfected with MYCN antisense RNA constructs. Concomitant with MYCN down-regulation, the level of MRP expression was decreased in the NBAS-4 and NBAS-5 antisense transfectants. These cells demonstrated significantly increased sensitivity to the high affinity MRP substrates vincristine, doxorubicin, sodium arsenate and potassium antimony tartrate, but not to the poor MRP substrates, taxol or cisplatin. Similarly, transfection of full-length MYCN cDNA into SH-EP neuroblastoma cells resulted in increased MRP expression and significantly increased resistance specifically to MRP substrates. The results provide evidence for the MYCN oncogene influencing cytotoxic drug response via regulation of MRP gene expression. Our data also provide a link between the malignant and chemoresistant phenotypes of this childhood malignancy.