MYCN Levels Research Articles

Abstract 2753: Targeting bromodomain and extra-terminal (BET) family member BRD4 for treatment of pediatric sarcomas.

Abstract Background. Potent inhibitors of histone binding modules have the potential to suppress, selectively, transcription of specific genes, including MYC family of transcription factors. JQ1 binds competitively to acetyl-lysine recognition motifs, or bromodomains. JQ1 inhibits BET bromodomain, and down-regulates MYC transcription. Ewing sarcoma (EWS) and alveolar rhabdomyosarcoma (ARMS) are associated with high expression of c-MYC and MYCN, respectively (Delmore JE, Cell 2011; Filippakopoulos P, Nature 2010). Consequently, we have evaluated sensitivity of EWS and ARMS cell lines and xenograft models to JQ1. Methods. EWS and ARMS cells were incubated with JQ1, or JQ1R (non-active enantiomer) for 96 hrs. Cell viability was determined by Alamar Blue staining. C-MYC and MYCN were determined by immunoblotting. Xenograft tumor lines were grown in CB17 female SCID mice. JQ1 was administered P.O. at 30 mg/kg (BID) or 50 mg/kg (SID) for up to 3 weeks. Assessment of JQ1 activity was as described (Houghton P, Ped Blood & Cancer, 2007). The effect of JQ1 on proliferation and tube formation of human umbilical vascular endothelial cells (HUVECs) was as described (Bid H, Mol Cancer Ther, 2012). Results. For all cell lines, the IC50 concentration for JQ1R (inactive enantiomer) was > 10 microM. EWS cell lines (n=9) segregated into two distinct groups with IC50 < 1 microM, (ES-1, ES-2, ES-3, ES-4, ES-7, ES-8) and cell lines with significantly higher IC50s (IC50 > 7 microM ES-6, EW8, CHLA-258). Seven of 9 ARMS cell lines demonstrated sensitivity to JQ1 (IC50 < 1 microM) whereas 2 lines were insensitive (IC50 > 10 microM Rh18, Rh30). JQ1 (500 nM, 24 hr) markedly down-regulated c-MYC in Rh41, and to a lesser extent in Rh36. MYCN expression in Rh5 ARMS cell line was also suppressed by JQ1. Of interest, JQ1 did not suppress c-MYC in any EWS cell line. In vivo, BID and SID dosing was well tolerated (mortality 1.2%) and gave similar antitumor activity. JQ1 significantly retarded growth of all 4 sarcoma models (Rh10, Rh28 ARMS and EW5, EW8 EWS lines. T/C at day 21 range 0.30 – 0.43). Of note, while EW8 cells in vitro were resistant to JQ1 (IC50 > 10 microM), the xenografts were equally sensitive as other lines to JQ1 treatment, suggesting JQ1 exerted effects on stroma/angiogenesis. In vitro, JQ1 (500 nM) suppressed tube formation of HUVECs without decreasing viability. Conclusions. JQ1 potently inhibited proliferation of EWS and ARMS cells in vitro, with distinct groups demonstrating either sensitivity or resistance. Sensitivity was associated with decreased levels of MYCN or c-MYC in some cell lines. In vivo, JQ1 demonstrated significant antitumor activity in 4 xenograft lines, including EW8 derived from cells resistant to JQ1 in vitro. The potential for JQ1 to exert anti-angiogenic effects is suggested by its potent inhibition of HUVECs differentiation. Supported through a grant from SWOG Citation Format: Linlin Xiao, Doris A. Phelps, Laurence A. Baker, Jun Qi, Hemant K. Bid, Peter J. Houghton. Targeting bromodomain and extra-terminal (BET) family member BRD4 for treatment of pediatric sarcomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2753. doi:10.1158/1538-7445.AM2013-2753

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

LIN28B Is a Neuroblastoma Oncogene

Abstract LIN28B drives neuroblastoma through suppression of let-7, which elevates MYCN levels.

FAK is a Critical Regulator of Neuroblastoma Liver Metastasis

Neuroblastomas express increased levels of gastrin-releasing peptide receptor (GRP-R). However, the exact molecular mechanisms involved in GRP-R-mediated cell signaling in neuroblastoma growth and metastasis are unknown. Here, we report that focal adhesion kinase (FAK), as a critical downstream target of GRP-R, is an important regulator of neuroblastoma tumorigenicity. We found that FAK expression correlates with GRP-R expression in human neuroblastoma sections and cell lines. GRP-R overexpression in SK-N-SH cells increased FAK, integrin α3 and β1 expressions and cell migration. These cells demonstrated flatter cell morphology with broad lamellae, in which intense FAK expression was localized to the leading edges of lamellipodia. Interestingly, FAK activation was, in part, dependent on integrin α3 and β1 expression. Conversely, GRP-R silencing decreased FAK as well as Mycn levels in BE(2)-C cells, which displayed a denser cellular morphology. Importantly, rescue experiments in GRP-R silenced BE(2)-C cells showed FAK overexpression significantly enhanced cell viability and soft agar colony formation; similarly, FAK overexpression in SK-N-SH cells also resulted in increased cell growth. These effects were reversed in FAK silenced BE(2)-C cells in vitro as well as in vivo. Moreover, we evaluated the effect of FAK inhibition in vivo. FAK inhibitor (Y15) suppressed GRP-induced neuroblastoma growth and metastasis. Our results indicate that FAK is a critical downstream regulator of GRP-R, which mediates tumorigenesis and metastasis in neuroblastoma.

Inhibition of cyclin-dependent kinase 1–induced cell death in neuroblastoma cells through the microRNA-34a–MYCN–survivin pathway

Neuroblastoma is the most common pediatric solid extracranial malignancy. Increased expression of MYCN, a member of Myc family, is associated with a poor prognosis in patients with neuroblastoma. Cyclin-dependent kinase 1 (CDK1) is known to be critical to the survival of Myc-overexpressing neoplasms. In this study, we investigated the effects of CDK1 inhibition on the viability of neuroblastoma cells and the underlying molecular mechanisms. We assessed the effects of CDK1 inhibitors on the viability of neuroblastoma cells and explored MYCN-associated mechanisms by using real-time reverse-transcription polymerase chain reaction, chromatin immunoprecipitation, and micro-RNA quantification. CDK1 inhibitors induced cytotoxicity and apoptosis and decreased expression of MYCN and survivin in neuroblastoma cells. Knockdown of MYCN mRNA expression caused significant cell death and transcomplementation of MYCN increased cell viability in neuroblastoma cells treated with CDK1 inhibitors. Furthermore, MYCN mRNA was inhibited by CDK1 inhibitor at the posttranscriptional level. The expression of survivin was also responsive to the change of MYCN levels, and according to the chromatin immunoprecipitation assay, MYCN bound the promoter region of the survivin gene. CDK1 inhibition caused elevation of microRNA-34a (miR-34a), and anti-miR-34a not only restored the expression of MYCN but also increased cell survival of neuroblastoma cells treated with CDK1 inhibitor. CDK1 inhibition-induced cell death was dependent, although not completely, on the decreased level of MYCN- and miR-34a-mediated, CDK1 inhibition-induced down-regulation of MYCN, which thereby decreased the transcriptional activation of MYCN on the survivin promoter. Wepropose that CDK1 inhibition-induced cell death of neuroblastoma cells occurs through the miR-34a-MYCN-survivin pathway.

Abstract 202: Expressional alterations in functional ultra-conserved non-coding RNAs in response to ATRA-induced differentiation and changes in MYCN levels in neuroblastoma cells

Abstract Neuroblastoma (NB) is a childhood cancer arising from precursor cells of the sympathetic nervous system with highly heterogeneous clinical behaviour, ranging from spontaneous regression to rapid progression and death due to disease. Amplification and over-expression of the MYCN transcription factor in NB tumors is highly correlated with poor survival. Children with high risk NB are given the synthetic retinoic acid, 13-cis-retinoic acid to reduce minimal residual disease, and exposure of a number of NB cell lines to the compound all-trans-retinoic acid (ATRA) induces neural differentiation along with the down-regulation of MYCN. Thus, identification of targets regulated by either ATRA or MYCN is of potential interest for the development of novel therapeutics. Ultra-conserved regions (UCRs) are sequences ≥200bp in length that are 100% conserved between human, mouse and rat genomes. In NB, transcribed UCRs (T-UCRs) exhibit specific profiles in MYCN-amplified (MNA) compared to non-MNA tumors (Mestdagh et al., Oncogene 2010 29:3583), and in high risk versus low risk tumors (Scaruffi et al., BMC Cancer 2009 9;441). Here we identify T-UCRs that are responsive to ATRA as well as T-UCRs that are directly or indirectly regulated by MYCN, and evaluate the biological significance of selected T-UCRs in functional studies of NB cell lines. We designed a custom tiling microarray to profile the expression of 481 UCRs in sense and antisense orientation (962 potential T-UCRs) in three ATRA-sensitive NB cell lines, and in SHEP-21N cells which contain a MYCN trans-gene controlled by a tetracycline responsive repressor element. Thirty-two T-UCRs were differentially expressed following ATRA treatment across all three ATRA sensitive cell lines (p<0.05), while analysis of doxycyclin-treated SHEP-21N cells (to deplete MYCN levels) indicated that 42 T-UCRs were altered by changing MYCN levels. In addition we identified a small number of T-UCRs, including T-UC.396 (intergenic) and T-UC.324 (antisense to host gene), whose expression was altered in both systems. These T-UCRs may represent independent ncRNAs directly or indirectly regulated by MYCN. Functionality of T-UC.300A and T-UC.324 was assessed by siRNA knockdown. While knockdown of T-UC.324 had no effect on cell viability or invasion, depletion of T-UC.300A resulted in a significant decrease in both cell viability and invasion. Our results indicate that significant numbers of T-UCRs have altered expression levels in response to changing MYCN levels and ATRA-induced differentiation. While the precise roles that these ncRNAs might play in cancer or in normal development are largely unknown, we have identified a T-UCR, T-UC.300A, whose down-regulation results in the decreased viability and invasiveness of ATRA-responsive cell lines and may play a functional role in ATRA-induced differentiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 202. doi:1538-7445.AM2012-202

C-MYC and SIRT1 locked in a vicious cycle

The c-MYC proto-oncogene represents a nodal point of gene regulation, as it integrates numerous mitogenic incoming signals and converts them into enhanced synthesis of the c-MYC transcription factor, which orchestrates the expression of hundreds of genes [1]. Deregulation of c-MYC, as it is found in ~50% of all human cancers, not only induces cellular proliferation and growth but also leads to activation of the p53 tumor suppressor protein [2], which mediates apoptosis and/or senescence. This intrinsic fail-safe mechanism prevents expansion of cells harboring dangerous oncogenic mutations. The necessity to bypass this barrier is thought to explain why p53 is frequently mutated in human cancers of all types. However, ectopic expression of c-MYC may immortalize certain primary cells in the presence of wild-type p53, suggesting that c-MYC circumvents the action of p53 by alternative mechanisms. We hypothesized that c-MYC may activate a factor, which abrogates or down-regulates p53 function directly. An attractive candidate for this function was SIRT1, an NAD+-dependent deacetylase, which has been shown to inhibit the function of p53 [3]. Recently, four papers showed that c-MYC and SIRT1 regulate each other via feedback loops [4-7]. The three more recent publications suggest the existence of a positive feedback loop between c-MYC and SIRT1 [4-6], whereas a study published earlier entertains a negative feedback [7]. The positive feedback suggests an oncogenic role of SIRT1, whereas the negative feedback implies a tumor suppressive activity of SIRT1. In our hands activation of conditional c-MYC alleles resulted in a robust increase in SIRT1 protein levels and activity. This was mediated on one hand via the direct induction of the NAMPT gene, which encodes nicotinamide phosphoribosyltransferase, the rate limiting enzyme of the NAD+ salvage pathway. The resulting increased NAD+ levels are known to mediate the activation of SIRT1 [8]. A second mode of SIRT1 activation was through sequestration of the SIRT1 inhibitor DBC1 (deleted in breast cancer 1) by c-MYC. In c-MYC-immortalized cells the increased levels and enzymatic activity of SIRT1 were necessary to prevent senescence and c-MYC-induced apoptosis. Furthermore, c-MYC is bound and deacetylated by SIRT1 which resulted in reduced K48- and increased K63-ubiquitin linkage, and ultimately increased c-MYC stability and activity. In cancer cells deregulation of this reciprocal activation between c-MYC and SIRT1 presumably contributes to evasion of senescence and apoptosis [6]. Increased expression of SIRT1 mRNA in cells with elevated c-MYC or N-MYC levels suggested that c-MYC may transactivate the SIRT1 gene [5, 7]. Although we could not detect an increase in SIRT1 mRNA after c-MYC activation [6], a concomitant induction of SIRT1 mRNA and post-transcriptional activation of the SIRT1 enzyme by c-MYC, as described above, may act synergistically. In the three studies describing a positive feedback, this involves a direct physical interaction between c-MYC and SIRT1, leading to increased c-MYC function [4-6]. Only Yuan et al. observed that the interaction with SIRT1 reduced c-MYC stability and inhibited c-MYC functions, such as transformation and transactivation [7]. The region of MYC interacting with SIRT1 was reported to be either the C-terminus of c-MYC [4] or MYC-Box I within the N-terminus of N-MYC [5]. Although three studies document deacetylation of c-MYC as a result of the SIRT1/c-MYC interaction, there are considerable discrepancies with respect to the functional consequences of c-MYC deacetylation. In agreement with our own observations obtained for c-MYC, Marshall et al. report that the N-MYC protein is stabilized in the presence of SIRT1. However, this is mediated indirectly through a novel transcriptional repressor complex consisting of N-MYC and SIRT1, which down-regulates expression of mitogen-activated protein kinase phosphatase 3 (MKP3) and thereby increases ERK-mediated phosphorylation of N-MYC at serine 62. Mao et al. did not observe significant effects of SIRT1 on c-MYC protein levels. Furthermore, they found that SIRT1 binds to the C-terminus of c-MYC, which mediates heterodimerization with MAX. Interestingly, SIRT1 expression increased the c-MYC/MAX association and also enhanced c-MYC transcriptional activity. Taken together, the majority of the reported analyses (3 of 4) describe a positive feedback between c-MYC and SIRT1. Multiple mechanisms of c-MYC activation seem to be involved in this feedback. Indeed, the SIRT1-mediated increase in c-MYC expression and function by enhanced ubiquitin 63 linkage, serine 62 phosphorylation and c-MYC/MAX heterodimerization may occur in parallel and act synergistically. In conjunction with the inactivation of p53 and/or other tumor suppressive SIRT1 substrates, SIRT1 therefore seems to act pro-tumorigenic at least in the context of cells and tumors, which display alterations in c-MYC-activating pathways or c-MYC itself.

Abstract 3139: The microRNA 17-92 Cluster Mediates Sonic Hedgehog-driven Neurogenesis In Ischemic Neural Progenitor Cells

Background: The Sonic hedgehog (Shh) pathway regulates stroke-induced neurogenesis. The present study investigated the functional role of the microRNA 17-92 (miR17-92) cluster in this process. Methods and Results: Analysis of miRNA microarray and real-time RT-PCR revealed that stroke substantially increased levels of individual members of the miR17-92 cluster: miR-18a (1.8±0.3), miR-19a (2.5±0.4), and miR-92a (1.9±0.3) expression in neural progenitor cells (NPCs) harvested from the subventricular zone (SVZ) of ischemic rats (n=6). Overexpression of the miR17-92 cluster in cultured NPCs significantly increased NPC proliferation measured by the number of BrdU positive cells (52±4% vs 28±2% in empty vector, n=3/group, p<0.05). Concurrently, overexpression of the miR17-92 cluster reduced PTEN (phosphatase and tensin homolog), a target of the miR17-92 cluster, protein levels by 70% compared to levels in NPCs transfected with an empty vector. PTEN suppresses cell proliferation. These data suggest that the stroke-upregulated miR17-92 cluster enhances NPC proliferation via downregulation of PTEN. To examine whether Shh regulates miR17-92 cluster expression, NPCs were incubated with recombinant human Shh (rhShh, 100ng/ml). We found that rhShh significantly (p<0.05) increased levels of individual members of the miR17-92 cluster: miR-18a (2.1±0.1), miR-19a (1.3±0.7), miR-19b (1.5±0.6) and miR-92a (1.9±0.8). Blockage of a Shh receptor Smo with cyclopamine suppressed rhShh-increased levels of miR-18a (0.9±0.08), miR-19a (0.7±0.01), miR-19b (0.6±0.05) and miR-92a (0.8±0.04). Attenuation of endogenous Shh in NPCs with siRNA also substantially decreased levels of miR-18a (0.6±0.1), miR-19a (0.4±0.05) and miR-92a (0.6±0.1) compared with levels in NPCs transfected with scrambled siRNA (1.0±0.2, n=3), indicating that Shh regulates miR17-92 expression. MYC is a downstream target of Shh. Western blots showed that stroke increased the protein level of N-MYC 1.8 fold in SVZ tissues and incubation of NPCs with rhShh elevated N-MYC levels by 1.8 fold, which was abrogated by cyclopamine (1.3 fold). N-MYC transduction resulted in significant increases in expression of the primary miR17-92 cluster (2.1±0.3 vs 1.0±0.2 in control group, n=3, p<0.05). These data suggest that the Shh pathway recruits N-MYC to regulate miR17-92 cluster expression in NPCs. Conclusion: Our data suggest a functional role of the miR17-92 cluster in mediating stroke-induced neurogenesis by the SHH/MYC signaling pathway, which provides new insight into molecular mechanisms of stroke-induced neurogenesis.

Antitumor Activity of Sustained N-Myc Reduction in Rhabdomyosarcomas and Transcriptional Block by Antigene Therapy

Rhabdomyosarcomas are a major cause of cancer death in children, described with MYCN amplification and, in the alveolar subtype, transcription driven by the PAX3-FOXO1 fusion protein. Our aim was to determine the prevalence of N-Myc protein expression and the potential therapeutic effects of reducing expression in rhabdomyosarcomas, including use of an antigene strategy that inhibits transcription. Protein expression was assessed by immunohistochemistry. MYCN expression was reduced in representative cell lines by RNA interference and an antigene peptide nucleic acid (PNA) oligonucleotide conjugated to a nuclear localization signal peptide. Associated gene expression changes, cell viability, and apoptosis were analyzed in vitro. As a paradigm for antigene therapy, the effects of systemic treatment of mice with rhabdomyosarcoma cell line xenografts were determined. High N-Myc levels were significantly associated with genomic amplification, presence of the PAX3/7-FOXO1 fusion genes, and proliferative capacity. Sustained reduction of N-Myc levels in all rhabdomyosarcoma cell lines that express the protein decreased cell proliferation and increased apoptosis. Positive feedback was shown to regulate PAX3-FOXO1 and N-Myc levels in the alveolar subtype that critically decrease PAX3-FOXO1 levels on reducing N-Myc. Pharmacologic systemic administration of the antigene PNA can eliminate alveolar rhabdomyosarcoma xenografts in mice, without relapse or toxicity. N-Myc, with its restricted expression in non-fetal tissues, is a therapeutic target to treat rhabdomyosarcomas, and blocking gene transcription using antigene oligonucleotide strategies has therapeutic potential in the treatment of cancer and other diseases that has not been previously realized in vivo.

Abstract B9: Critical Early microRNA-Mediated Signalling Events During ATRA Induced Neuroblastoma Cell Differentiation

Abstract Introduction: Neuroblastoma (NB) is a childhood cancer arising from precursor cells of the sympathetic nervous system with highly heterogeneous clinical behavior, ranging from spontaneous regression to rapid progression and death due to disease. Amplification and over-expression of the MYCN transcription factor in neuroblastoma tumors is highly correlated with poor survival of patients. Children with high risk NB are given the synthetic retinoic acid, 13-cis-retinoic acid, to reduce minimal residual disease, and exposure of a number of NB cell lines to the related compound, all-trans-retinoic acid (ATRA), induces neural differentiation along with the down-regulation of MYCN. We previously determined that miRNA-10a/b plays a critical role in the process of ATRA induced neuroblastoma cell differentiation through direct targeting of nuclear co-repressor receptor 2 (NCOR2) (Foley et al Cell Death & Differentiation 2011: 18:1089). Here, we have profiled three ATRA sensitive (SK-N-BE, LAN5 and SHSY-5Y) and two ATRA resistant (Kelly and SK-N-AS) neuroblastoma cell lines for the expression of 750 miRNA loci at early (3 hours) and late (7 days) time points following ATRA treatment. The purpose of our experiments was to identify critical early miRNA signaling events, as well as miRNAs that might mediate sensitivity versus resistance to ATRA. Methods: All cells were treated with 5 μmol ATRA (Sigma, St. Louis, MO) which was continuously administered by replacing the medium every 24 hrs. MiRNA expression profiling was carried out using TaqMan Low Density Arrays from Applied BioSystems (Carlsbad, CA). Results: miR-10a, -34a, -125b, -28-5p, and -183 were consistently up-regulated and miR-450-5p, -151-3p and -138-2 were consistently down-regulated >2 fold (p < 0.05) in all three ATRA sensitive cell lines at three hours post-ATRA. At 7 days post-ATRA, there were more differentially expressed miRNAs, with 13 miRNAs being up-regulated and 18 down-regulated > 2 fold (p < 0.05) across all three cell lines. Among the miRNAs differentially expressed at 3 hours post-ATRA, miR-10a, miR-125b and miR-34a remained differentially expressed at 7 days post-ATRA. Remarkably, none of the differentially expressed miRNAs in the ATRA sensitive cell lines were differentially expressed in the ATRA resistant cell lines at the three hour time point, and very few displayed altered expression in the ATRA resistant cell lines even after a 7 day exposure to ATRA. qPCR and Western blot analysis of the critical miR-10a target, NCOR2, revealed that this gene is down regulated at both mRNA and protein level at the 3 hour time point. In addition to the targeting of NCOR2 by miR-10a, we noted that two additional up-regulated miRNAs, miR-125b and miR-132 (significantly up-regulated within 7 days post-ATRA) are also predicted to target NCOR2, indicating substantial redundancy in miRNA mediated signaling. Direct targeting of NCOR2 by these additional miRNAs was experimentally validated by luciferase reporter assays. This redundancy in miRNA targeting explains why antagomir inhibition of single miRNAs prior to ATRA treatment only delays, but does not block the induction of differentiation. Finally, we present evidence that an initial early decrease in MYCN levels, which is well known to occur in neuroblastoma cells treated with ATRA, is miRNA mediated. Conclusions: We have identified critical and redundant early miRNA signaling events that take place during the process of ATRA induced neuroblastoma cell differentiation. The absence of these events in ATRA resistant cell lines suggests new avenues of investigation for elucidating potential miRNA mediated mechanisms involved with ATRA therapy failure. Citation Format: Niamh H. Foley, Isabella M. Bray, Raquel Fernandez, Raymond L. Stallings. Critical Early microRNA-Mediated Signalling Events During ATRA Induced Neuroblastoma Cell Differentiation [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr B9.

Metastatic neuroblastoma of the mandible: a cytogenetic and molecular genetic study

Neuroblastoma (NB) jaw metastases are rare. Here, we report on cytogenetic and genetic studies on metastatic NB to the mandible. A 7-year-old boy, with an abdominal neuroblastoma, presented with a mass of the left body of the mandible. Cytogenetic analysis of the original tumor and the mandibular lesion biopsies revealed similar heterogenous subclones with 42 ~ 47,XY,+der(1)(q11 → qter),-2,del(7)(q21.1 → qter),-8,-9,-10,-11,del(11)(q13.3 → qter),-13,-14,-15,-17, + 18-18,der(18)(?),+21,+m1,+m2,+m3,+m4,+m5,+m6,+m7[cp25]. The different markers were identified by SKY analysis. Most of the cells carried 3-6 of these translocations: der(1;21), der(2;9;17), der(2;15;18), der(2;15;Y), der(8;10), der(10;17). Molecular examination using Neuroblastoma MLPA kit (MRC-Holland) revealed gain of 1q25, 1q42, 2q33, 2p23, 2p24 (N-myc), and 21q22, and loss of 11q22, 11q23, 17p13, and 17q11. FISH analysis using N-myc probe showed high amplification levels of N-myc. The cytogenetic and molecular genetic work-ups revealed that the mandibular lesion is a metastasis of the original abdominal tumor and not a second primary caused by the aggressive treatment. Clinical parameters such as : patient's age, site of primary tumor and the mandibular metastasis, together with poor prognosis genetic markers explain the patient's short-term survival.

Differential Regulation of N-Myc and c-Myc Synthesis, Degradation, and Transcriptional Activity by the Ras/Mitogen-activated Protein Kinase Pathway

Myc transcription factors are important regulators of proliferation and can promote oncogenesis when deregulated. Deregulated Myc expression in cancers can result from MYC gene amplification and translocation but also from alterations in mitogenic signaling pathways that affect Myc levels through both transcriptional and post-transcription mechanisms. For example, mutations in Ras family GTPase proteins that cause their constitutive activation can increase cellular levels of c-Myc by interfering with its rapid proteasomal degradation. Although enhanced protein stability is generally thought to be applicable to other Myc family members, here we show that c-Myc and its paralog N-Myc respond to oncogenic H-Ras (H-Ras(G12V)) in very different ways. H-Ras(G12V) promotes accumulation of both c-Myc and N-Myc, but although c-Myc accumulation is achieved by enhanced protein stability, N-Myc accumulation is associated with an accelerated rate of translation that overcomes a surprising H-Ras(G12V)-mediated destabilization of N-Myc. We show that H-Ras(G12V)-mediated degradation of N-Myc functions independently of key phosphorylation sites in the highly conserved Myc homology box I region that controls c-Myc protein stability by oncogenic Ras. Finally, we found that N-Myc and c-Myc transcriptional activity is associated with their proteasomal degradation but that N-Myc may be uniquely dependent on Ras-stimulated proteolysis for target gene expression. Taken together, these studies provide mechanistic insight into how oncogenic Ras augments N-Myc levels in cells and suggest that enhanced N-Myc translation and degradation-coupled transactivation may contribute to oncogenesis.

Dickkopf‐3 is regulated by the MYCN‐induced miR‐17‐92 cluster in neuroblastoma

Neuroblastoma (NB) is a paediatric tumour with a remarkable diverse clinical behaviour. Approximately half of the high stage aggressive tumours are characterized by MYCN gene amplification but our understanding of the role of MYCN in NB oncogenesis is incomplete. Previous studies have shown that MYCN expression is inversely correlated with expression of Dickkopf-3 (DKK3), a gene encoding an extracellular protein with presumed tumour suppressor activity, but direct MYCN regulation of DKK3 was excluded leaving the mechanism of regulation unexplained. Given the recently established role of MYCN-regulated miRNAs in downregulation of protein-coding genes and predicted seeds for miR-17-92 cluster members within the DKK3 3'UTR, we hypothesized that this mechanism would act in MYCN regulation of DKK3. To investigate this, we used a validated miR-17-92-inducible cellular system and could demonstrate robust downregulation of DKK3 mRNA and protein levels upon miR-17-92 overexpression. Next, two of the three predicted miRNAs, miR-19b and miR-92a, were shown to lower DKK3 protein levels, in addition to measurable DKK3 mRNA knock-down by miR-92a. Direct interaction between miR-19b or miR-92a and the 3'UTR of DKK3 was validated using luciferase reporter assays. In conclusion, this study demonstrates that the MYCN-induced downregulation of DKK3 results from direct upregulation of miR-17-92 components effecting both DKK3 mRNA stability and translation which further contributes to the pleiotropic oncogenic effect of elevated MYCN levels. The strict MYCN-mediated regulation of DKK3 is suggestive for an important downstream function of the MYCN protein and thus warrants further investigations to unravel the role of DKK3 in NB.

Effects of small molecule inhibitors of PI3K/Akt/mTOR signaling on neuroblastoma growth in vitro and in vivo

Activation of the PI3K/Akt signaling pathway is correlated with poor prognosis in neuroblastoma, the most common and deadly extracranial tumor of childhood. In this study, we show that the small-molecule inhibitors of phosphoinositide-dependent protein kinase-1 (PDK1) OSU03012 and the dual class I PI3K/mTOR inhibitor PI103 have profound effects on neuroblastoma survival in vitro and in vivo. Both OSU03012 and PI103 inhibited neuroblastoma growth in vitro. In treated cells, OSU03012 induced apoptosis and an S phase cell cycle arrest, whereas only minor apoptosis was detected in PI103 treated cells together with a G1 arrest. Both OSU03012 and PI103 downregulated phosphorylation of Akt and inhibited the downstream targets glycogen synthase kinase-3β (GSK3β) and p70 S6 kinase-1 (S6K1), as well as downregulated the expression of cyclin D1 and Mycn protein. Neuroblastoma cells expressing high levels of Mycn were more sensitive to OSU03012 or PI103 compared with cells expressing low Mycn levels. Both compounds significantly inhibited the growth of established, subcutaneous MYCN-amplified neuroblastoma xenografts in nude NMRI nu/nu mice. These results suggest that inhibition of the PI3K/Akt signaling pathway represent a clinical relevant target for the treatment of patients with high-risk MYCN-amplified neuroblastoma.

MYCN and MYC regulate tumor proliferation and tumorigenesis directly through BMI1 in human neuroblastomas

The BMI1 gene is overexpressed in ≈ 90% of human neuroblastomas. However, little is known about the regulation of BMI1 expression. Using microarray and immunohistochemical analysis, we show that BMI1 expression correlated with MYCN levels in MYCN-amplified human neuroblastomas, and with MYC levels in the MYCN-nonamplified group. We further demonstrated that BMI1 is a direct target gene of MYCN/MYC in 3 neuroblastoma cell lines: BE (2)-C, LAN1, and SH-SY5Y. Overexpression of MYCN or MYC transactivated the BMI1 promoter and up-regulated BMI1 gene expression. shRNA-mediated knockdown of MYCN or MYC decreased BMI1 gene expression. Chromatin immunoprecipitation and point-mutation assays revealed that both MYCN and MYC bind to the E-box within the BMI1 promoter. Overexpression of BMI1, MYCN, and MYC independently increased both cell proliferation and tumor growth. Conversely, specific inhibition of BMI1, MYCN, and MYC decreased tumor cell proliferation and tumor growth. Interestingly, BMI1 suppression in MYCN/MYC-overexpressing cells resulted in significantly greater inhibition compared to that in mock-transduced and parental cells. Our results indicate that MYCN and MYC regulate BMI1 gene expression at the transcriptional level and that dysregulation of the BMI1 gene mediated by MYCN or MYC overexpression, confers increased cell proliferation during neuroblastoma genesis and tumor progression.

Bmi1 puSHHes reprogramming

In 2006, the group of Shinya Yamanaka demonstrated that somatic cells could be reprogrammed into induced pluripotent stem cells (iPSCs) by ectopic expression of four transcription factors associated to stemness: Oct4, Sox2, Klf4 and c-Myc 1. This groundbreaking discovery opened the possibility of generating patient-specific cells for research, drug development and regenerative medicine. Due to the tremendous potential of its clinical applications, understanding the process of reprogramming has become a priority and one of the most fascinating biomedical research topics. In the last five years, scientists around the world are racing to solve two main issues regarding reprogramming. The first one is technical and consists in improving the efficiency of reprogramming and avoiding the necessity to integrate exogenous DNA into the reprogrammed host cells. Several advances have been made over the last years, both in replacing individual factors with chemicals, and in eliminating the need to alter the genome of the host cell 2. However, pure chemical reprogramming has not yet been achieved despite intense efforts, and integration-free reprogramming is still poorly efficient 2. Another major focus of research is to understand the molecular mechanisms underlying reprogramming. However, the low efficiency of the process makes this task challenging. Recently, our group reported that the Ink4a/Arf locus is a major barrier for reprogramming 3. For example, complementation of the Yamanaka factors with an shRNA against the Ink4a/Arf locus greatly improves reprogramming efficiency 3. Bmi1 is part of the Polycomb Repressive Complex 1 (PRC1) involved in transcriptional silencing through heterochromatinization. The Bmi1 gene is a well-characterized oncogene that silences the Ink4a/Arf locus and cooperates with c-Myc in lymphomagenesis 4 and it is overexpressed in several human cancers 5. In addition to this, Bmi1 has been shown over the years to be a key regulator of self-renewal in multiple adult stem cells including hematopoietic stem cells (HSCs), neural stem cells (NSCs) and, more recently, prostate stem cells (PrSCs) 5, 6. Sonic hedgehog (Shh) signaling is a major developmental signaling pathway and Bmi1 together with N-Myc and cyclin D2 are major downstream effectors of Shh for proliferation and self-renewal of cerebellar progenitor cells 5. Based on the impact of Bmi1 on the Ink4a/Arf locus and on stem cells, one might speculate what is the role of Bmi1 in reprogramming? In a recent paper published in Cell Research, Moon et al. 7 explore the role of Bmi1 in reprogramming. When overexpressing Bmi1 in mouse embryonic fibroblasts (MEFs), the authors show a marked increase in the levels of endogenous Sox2, N-Myc and Klf4, together with drastically reduced levels of Ink4a, Arf and p53. In line with this, Moon et al. demonstrate that iPSCs can be generated from MEFs using a simplified reprogramming cocktail consisting only of Bmi1 and Oct4, and with comparable efficiency to the Yamanaka three-factor cocktail (Sox2, Klf4 and Oct4). This is an important and satisfying finding, however, the oncogenic activity of Bmi1 is not very appealing for future therapeutic applications. Therefore, Moon et al. took their findings one step further. As mentioned above, the Shh signaling pathway was known to induce Bmi1 expression in neural progenitors 5. They treated MEFs with Shh and exposed them to NSC culture medium. Using this protocol, MEFs were transdifferentiated into NSC-like cells, a process that was also recapitulated by overexpression of Bmi1 7. Neural stem cells were previously known for being reprogrammed only with Oct4 due to their high endogenous levels of Sox2 and Klf4 8. Therefore, Moon et al. transduced Oct4 into the Shh-induced NSC-like cells and were able to obtain iPSCs. This takes the authors to their last tour de force by which they reprogram MEFs with the combination of two chemical Shh agonists (oxysterol and purmorphamine) and Oct4 7. The above findings illuminate the process of reprogramming and also get us closer to chemical reprogramming. The advantage of a hypothetical chemical reprogramming resides in its simplicity and safety, which are of high importance for future therapeutic applications. Previously, two other teams have published in Cell Research that combining Oct4 with either BMPs or with a combination of small molecules are sufficient to reprogram somatic cells to iPSCs 9, 10. The new findings by Moon et al. 7 broaden the spectrum of molecules that could be used in combination with Oct4. So far, Oct4 is the only factor that has not been replaced by small molecules. However, based on the speed of progress in this field, it is not unrealistic to anticipate that pure chemical reprogramming will be achieved in the near future.

A Genome-Wide Search for Promoters That Respond to Increased MYCN Reveals Both New Oncogenic and Tumor Suppressor MicroRNAs Associated with Aggressive Neuroblastoma

MYCN is a major driver of neuroblastoma tumorigenesis and MYCN amplification is the worst prognostic indicator of aggressive NB. To identify potentially therapeutic tumor suppressor microRNAs for aggressive NB, we utilized a conditional MYCN system to simulate MYCN-amplified and nonamplified tumor types and performed a genome-wide search for MYCN target microRNA promoters differentially repressed under high MYCN conditions. We identified 20 gene promoters hosting 30 microRNAs that were directly bound and differentially regulated by MYCN. Eleven of these genes showed significant clinical correlations for neuroblastoma with 4 genes linked with better survival and 7 genes linked with poor survival. Surprisingly, expression analysis of host genes and microRNAs demonstrated that 8 of 11 pairs were repressed by high levels of MYCN regardless of the clinical correlation of the host gene. We therefore predicted these intronic microRNAs would be tumor suppressors. In fact, detailed gain of function studies for two miRs, miR-591 and miR-558, confirmed potent tumor suppressive effects for miR-591 in orthotopic neuroblastoma xenografts. However, miR-558 markedly increased colony formation, proliferation, and tumor growth in vivo. Our data reveal host-gene independent functions of MYCN-target microRNAs and demonstrate that MYCN represses both tumor suppressive and proproliferative microRNAs.

Abstract LB-367: New evidence supports the potential use of methotrexate in children with MYCN-amplified neuroblastoma.

Abstract Background Folate is essential for cell growth and differentiation. Cancer cells frequently up-regulate folate receptors to increase folate uptake to meet their elevated need for nucleotides to support DNA synthesis and growth. SLC19A1 encodes for a major folate or anti-folate transporter and has been reported to be regulated by oncogenic transcription factors. In childhood neuroblastoma, amplification of the MYCN oncogene is a strong prognostic marker for predicting unfavourable outcome. In light of previous findings showing that overexpression of SLC19A1 increases folate influx and methotrexate (MTX) sensitivity, we aimed to investigate the relationship between SLC19A1 expression, MYCN amplification and expression, to determine if this relationship modified MTX sensitivity in neuroblastoma. Methods Expression levels of SLC19A1 and MYCN were examined in 42 neuroblastoma patient samples and in two independent in vitro MYCN functional systems. The impact of MYCN amplification/expression and SLC19A1 expression on MTX sensitivity were evaluated on 13 neuroblastoma cell lines. SLC19A1 G80A polymorphism was also examined to determine its impact on SLC19A1 gene expression and MTX sensitivity. Results Expression levels of SLC19A1 and MYCN were positively correlated in neuroblastoma tumour samples (p=0.0001). Induction of MYCN in vitro increased the mRNA expression levels of SLC19A1 by 12-fold (p=0.0003) while silencing of MYCN reduced the expression levels of SLC19A1 by 7-fold (p=0.0017). Neuroblastoma cell lines with MYCN overexpression or amplification were at least 2000-fold more sensitive to MTX than those without (expression: p=0.0002; amplification: p<0.0001). However, SLC19A1 G80A polymorphism did not appear to impact on SLC19A1 gene expression or MTX sensitivity. Conclusions Our data indicate a potential functional link between MYCN and the regulation of anti-folate uptake. Our findings suggest that MYCN amplification confers MTX sensitivity through the regulation of the folate receptor; SLC19A1. Targeting SLC19A1 mediated anti-folate uptake in patients with MYCN amplification may provide a new therapeutic approach in children with neuroblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-367. doi:10.1158/1538-7445.AM2011-LB-367

A SP1/MIZ1/MYCN Repression Complex Recruits HDAC1 at theTRKAandp75NTRPromoters and Affects Neuroblastoma Malignancy by Inhibiting the Cell Response to NGF

Neuroblastoma is the most common extracranial solid tumor of childhood. One important factor that predicts a favorable prognosis is the robust expression of the TRKA and p75NTR neurotrophin receptor genes. Interestingly, TRKA and p75NTR expression is often attenuated in aggressive MYCN-amplified tumors, suggesting a causal link between elevated MYCN activity and the transcriptional repression of TRKA and p75NTR, but the precise mechanisms involved are unclear. Here, we show that MYCN acts directly to repress TRKA and p75NTR gene transcription. Specifically, we found that MYCN levels were critical for repression and that MYCN targeted proximal/core promoter regions by forming a repression complex with transcription factors SP1 and MIZ1. When bound to the TRKA and p75NTR promoters, MYCN recruited the histone deacetylase HDAC1 to induce a repressed chromatin state. Forced re-expression of endogenous TRKA and p75NTR with exposure to the HDAC inhibitor TSA sensitized neuroblastoma cells to NGF-mediated apoptosis. By directly connecting MYCN to the repression of TRKA and p75NTR, our findings establish a key pathway of clinical pathogenicity and aggressiveness in neuroblastoma.

Combinatorial Regulation of Neuroblastoma Tumor Progression by N-Myc and Hypoxia Inducible Factor HIF-1α

In human neuroblastoma, amplification of the MYCN gene predicts poor prognosis and resistance to therapy. Because hypoxia contributes to aggressive tumor phenotypes, predominantly via two structurally related hypoxia inducible factors, HIF-1α and HIF-2α, we examined hypoxia responses in MYCN-amplified neuroblastoma cells. We demonstrate here that HIF-1α, but not HIF-2α, is preferentially expressed in both MYCN-amplified neuroblastoma cells and primary tumors in comparison to samples without MYCN amplification. Our results showed that interplay between N-Myc and HIF-1α plays critical roles in neuroblastoma. For example, high levels of N-Myc override HIF-1α inhibition of cell cycle progression, enabling continued proliferation under hypoxia. Furthermore, both HIF-1α and N-Myc are essential for the Warburg effect (aerobic glycolysis) in neuroblastomas by activating the transcription of multiple glycolytic genes. Of note, expressions of Phosphoglycerate Kinase 1 (PGK1), Hexokinase 2 (HK2), and Lactate Dehydrogenase A (LDHA) were each significantly higher in MYCN-amplified neuroblastomas than in tumors without MYCN amplification. Interestingly, MYCN-amplified neuroblastoma cells are "addicted" to LDHA enzymatic activity, as its depletion completely inhibits tumorigenesis in vivo. Thus, our results provide mechanistic insights explaining how MYCN-amplified neuroblastoma cells contend with hypoxic stress and paradoxically how hypoxia contributes to neuroblastoma aggressiveness through combinatorial effects of N-Myc and HIF-1α. These results also suggest that LDHA represents a novel, pharmacologically tractable target for neuroblastoma therapeutics.