MYCN Protein Levels Research Articles

Abstract 4345: AZD8055, a combined TORC1/TORC2 inhibitor regulates Mycn protein expression and prevents neuroblastoma growth in vitro and in vivo

Abstract Neuroblastoma is the most common and deadly solid tumour of childhood (Brodeur, 2003). Amplification of the MYCN oncogene occurs in 25% of NBs correlating with poor clinical outcome. High risk neuroblastoma responds to current therapies but rapidly acquires chemoresistance; therefore alternative treatment strategies are required. Because of the importance of MYCN amplification in high risk neuroblastoma and specific expression of Mycn protein in tumour tissue, we have examined indirect methods of destabilising Mycn by targeting key components of upstream signalling pathways, responsible for maintenance of Mycn protein levels. Mycn protein stability is maintained by the RAS (RAF/MEK/ERK) and PI3K (PI3K/AKT/mTOR) pathways. The mammalian target of rapamycin (mTOR) plays an important role in cell proliferation, metabolism and tumourigenesis, and is frequently altered in cancer. Rapamycin and its analogues target the TORC1 complex and are effective against tumour xenografts but are limited by consequent up regulation of PI3K activity. Inhibition of TORC1 alleviates a TORC1 dependent negative feedback loop, with resulting activation of PI3K and AKT-dependent survival pathways. AZD8055 is an mTOR kinase inhibitor that inhibits both TORC complexes with IC50 values in the nanomolar (nM) range. Treatment of N-myc wild-type or phosphomutant expressing neuroblastoma cell lines with AZD8055 inhibited mTOR-dependent phosphorylation of key pathway components, coincident with selective inhibition of wild-type but not phospho-mutated N-myc protein. Cell proliferation was reduced and sensitive cells accumulated in G1 as shown by flow cytometric analysis. Cells expressing N-myc phosphomutant proteins showed less sensitivity to AZD8055, with GI50 values above mechanistic range for targeted effects of this drug. The ability of AZD8055 to inhibit neuroblastoma tumour progression was tested in a transgenic model of neuroblastoma (TH-MYCN) that over expresses Mycn protein and is predisposed to spontaneous tumour formation (Weiss, 97). We present data showing that AZD8055 inhibits tumour progression, reduces tumour cell proliferation and targets the mTOR complex in vivo. In conclusion, inhibition of mTOR signalling is highly effective against MYCN-driven cell lines and tumours, and therefore could be a promising therapeutic strategy for targeted treatment of MYCN-amplified 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 4345. doi:10.1158/1538-7445.AM2011-4345

Abstract LB-142: Regulation of MYCN stability by reactive oxygen species in neuroblastoma

Abstract Neuroblastoma (NB) is the most common extracranial solid tumor in children. Amplification of the MYCN proto-oncogene is associated with older age, rapid tumor progression, and the worst outcome of this disease. As MYCN amplification leads to its over-expression, high-level expression of MYCN is thought to cause an aggressive behavior of MYCN amplified tumors. In fact, the forced reduction of MYCN protein expression by siRNA results in growth suppression and apoptosis of NB cells with MYCN amplification. We have identified several compounds that can rapidly destabilize the MYCN protein (within a few hours), in NB cells and cause growth suppression. The compounds include FCCP, OSU-03012, and Salinomycin. Our recent data suggest that a common effect of the above compounds appears to be the inhibition of mitochondrial functions. Moreover, ascorbic acid, an anti-oxidant, abolishes the effect of these compounds on MYCN protein stability. It is known that inhibition of mitochondrial oxidative phosphorylation increases the production of reactive oxygen species (ROS), which include superoxide (O2-), hydroxyl radical (•OH), and hydrogen peroxide (H2O2). Anti-oxidants are also known to quench ROS. Preliminary data showed that ROS was detected when the IMR5 NB cells were treated with OSU-03012 for three hours. In addition, we have found that forced over-expression of pVHL, an E3 ubiquitin ligase, in NB cell lines results in reduction of MYCN protein levels. We are currently testing whether ROS regulates MYCN stability by hydroxylation of proline residues on MYCN, and whether pVHL recognizes the ROS-modified MYCN, which is then subjected to rapid proteasome-dependent degradation. 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-142. doi:10.1158/1538-7445.AM2011-LB-142

Dexamethasone destabilizes Nmyc to inhibit the growth of hedgehog-associated medulloblastoma.

Mouse studies indicate that the synthetic glucocorticoid dexamethasone (Dex) impairs the proliferation of granule neuron precursors in the cerebellum, which are transformed to medulloblastoma by activation of Sonic hedgehog (Shh) signaling. Here, we show that Dex treatment also inhibits Shh-induced tumor growth, enhancing the survival of tumor-prone transgenic mice. We found that Nmyc was specifically required in granule cells for Shh-induced tumorigenesis and that Dex acted to reduce Nmyc protein levels. Moreover, we found that Dex-induced destabilization of Nmyc is mediated by activation of glycogen synthase kinase 3beta, which targets Nmyc for proteasomal degradation. Together, our findings show that Dex antagonizes Shh signaling downstream of Smoothened in medulloblastoma.

Transcriptional Repression of SKP2 Is Impaired in MYCN-Amplified Neuroblastoma

The cell cycle regulator, SKP2, is overexpressed in various cancers and plays a key role in p27 degradation, which is involved in tumor cell dedifferentiation. Little is known about the mechanisms leading to impaired SKP2 transcriptional control in tumor cells. We used neuroblastoma as a model to study SKP2 regulation because SKP2 transcript levels gradually increase with aggressiveness of neuroblastoma subtypes. The highest SKP2 levels are found in neuroblastomas with amplified MYCN. Accordingly, we found 5.5-fold (range, 2-9.5) higher SKP2 core promoter activity in MYCN-amplified cells. Higher SKP2 core promoter activity in MYCN-amplified cells is mediated through a defined region at the transcriptional start site. This region includes a specific E2F-binding site that makes SKP2 activation largely independent of mitogenic signals integrated through the SP1/ELK-1 site. We show by chromatin immunoprecipitation that SKP2 activation through the transcriptional start site in MYCN-amplified cells is associated with the low abundance of pRB-E2F1 complexes bound to the SKP2 promoter. Transcriptional control of SKP2 through this regulatory mechanism can be reestablished in MYCN-amplified cells by restoring pRB activity using selective small compound inhibitors of CDK4. In contrast, doxorubicin or nutlin-3 treatment-both leading to p53-p21 activation-or CDK2 inhibition had no effect on SKP2 regulation in MYCN-amplified cells. Together, this implies that deregulated MYCN protein levels in MYCN-amplified neuroblastoma cells activate SKP2 through CDK4 induction, abrogating repressive pRB-E2F1 complexes bound to the SKP2 promoter.

Abstract 4758: A new syngeneic MYCN-overexpressing neuroblastoma mouse model and MYCN-DNA vaccine

Abstract Neuroblastoma (NB) is the most common solid extracranial tumor in childhood. High-level expression of MycN caused by amplification of the gene plays an important role in maintaining the malignant phenotype of NB and is the most significant marker for poor prognosis overall. Recent studies suggested that MYCN is a suitable target for immunotherapy, but up to now, a syngeneic NB mouse model over expressing MYCN and is not available to examine immunotherapeutic strategies in vivo. Here, we report the development of a tetracycline inducible MYCN expressing murine NB cell line syngeneic to A/J mice and a new MYCN-DNA vaccine. The murine NB cell line C1300, syngeneic to A/J mice, was stably transfected with a tetracycline inducible MYCN expression vector system T-RExTM (Invitrogen). Stable transfection was verified by real-time PCR and Western-Blot, revealing significant high expression levels of MYCN RNA and protein, respectively. Characterization of the murine MYCN expressing cells in vitro and in vivo will be reported. Furthermore, a new MYCN-DNA vaccine, based on epitopes encoding for three peptides from the murine MYCN protein sequence with high affinity to the A/J mouse MHC class I allele H2-Kk, was designed and tested in vivo for its ability to induce an antigen-specific immune response. Lymphocytes isolated from A/J mice vaccinated with the MYCN minigen vaccine effectively lysed wild type C1300 cells in cytotoxicity assays in contrast to lymphocytes from control mice. Additionally, lymphocytes from minigene vaccinated mice produced significantly higher amounts of IFN-γ after stimulation with irradiated C1300 cells, than lymphocytes from control mice, indicating the induction of cytotoxic T lymphocytes by the vaccine that actively kill even parental C1300 tumor cells with relatively low MYCN expression. We suspect that this effect will be enhanced when the newly established high MYCN-expressing C1300 cells are employed in a similar cytotoxicity assay. Results to support this hypothesis will be reported at the meeting. In summary, we report the development of a new MYCN-DNA vaccine and a murine tetracycline inducible MYCN NB cell line providing an important new syngeneic mouse model for in vivo evaluation of new immunotherapeutic strategies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4758.

Abstract LB-387: MYCN dependence of tumor propagating cells in an orthotopic model of medulloblastoma

Abstract Medulloblastoma (MB) is the most common paediatric brain tumor and is a major cause of cancer death in children. MYCN expression is critical to cerebellar development, regulates the fate of neural progenitor cells (NPCs) and drives the expansion of tumor propagating cells (TPCs) thought to represent the cell-of-origin for this disease. To examine the role of MYCN in MB tumorigenesis, we established neurosphere cell lines enriched for putative TPCs from the cerebella of GTML mice (transgenic for doxycycline-regulable overexpression of MYCN and luciferase). These mice are predisposed to anaplastic/large-cell MB (AACR abstract LB-79). Neurospheres derived from GTML cerebella with positive luciferase imaging signals but no visible tumor tissue exhibited several characteristics consistent with enrichment for TPCs and/or NPCs. Spheroids proliferated efficiently in the absence of exogenous growth factor, exhibited self-renewal in clonal dilution assays, expressed high levels of Mycn protein and the NPC markers nestin and SOX2, and in response to serum, differentiated along neuronal and glial lineages (increased GFAP and III tubulin expression by immunofluorescence). Finally, orthotopic reimplantation at low titer (<250 cells) recapitulated anaplastic/large-cell MB tumor formation with short latency (<14 days) and complete penetrance. Suppression of MYCN with doxycycline (confirmed by loss of luciferase imaging) caused growth arrest via a G1 blockade of neurospheres in vitro. Pretreatment of implanted spheres significantly reduced tumor penetrance (<60%) and increased latency (2-3 months). Efficient regression of established tumors was observed with concomitant loss of nestin expression and increases in differentiated glial and neuronal markers. Taken together, these data suggest that MYCN expression drives MB tumor formation by maintenance or expansion of a population of relatively undifferentiatedTPCs present in normal cerebella. This model represents a significant tool for further investigation of the role of MYCN in MB initiation and progression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-387.

Abstract 568: Transcriptional repression of SKP2 is impaired in MYCN-amplified neuroblastoma

Abstract The cell cycle regulator, SKP2, is overexpressed in various cancers, and plays a key role in p27 degradation, which is involved in tumor cell dedifferentiation. Little is known about the mechanisms leading to impaired SKP2 transcriptional control in tumor cells. We used neuroblastoma as a model to study SKP2 regulation because SKP2 transcript levels gradually increase with aggressiveness of neuroblastoma subtypes. Highest SKP2 levels are found in neuroblastomas with amplified MYCN. Accordingly, we found 5.5-fold (range 2-9.5) higher SKP2 core promoter activity in MYCN-amplified cells. Higher SKP2 core promoter activity in MYCN-amplified cells is mediated through a defined region at the transcriptional start site (TSSR). This region includes a specific E2F-binding site that makes SKP2 activation largely independent of mitogenic signals integrated through the SP1/ELK-1 site. We demonstrate by chromatin immunoprecipitation that SKP2 activation through the TSSR in MYCN-amplified cells is associated with low abundance of pRB-E2F1 complexes bound to the SKP2 promoter. Transcriptional control of SKP2 via this regulatory mechanism can be re-established in MYCN-amplified cells by restoring pRB activity using selective small compound inhibitors of CDK4. In contrast, doxorubicin or nutlin-3 treatment - both leading to p53-p21 activation - or CDK2 inhibition had no effect on SKP2 regulation in MYCN-amplified cells. Together, this implies that deregulated MYCN protein levels in MYCN-amplified neuroblastoma cells activate SKP2 through CDK4 induction, abrogating repressive pRB-E2F1 complexes bound to the SKP2 promoter. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 568.

Importance of CDK7 for G1 Re-Entry into the Mammalian Cell Cycle and Identification of New Downstream Networks Using a Computational Method~!2009-11-05~!2010-03-01~!2010-04-02~!

Many of the key molecules in cell cycle progression (e.g. pRB, cyclin complexes) and their basic interactions are oncogene or tumor suppressor genes, which are well characterized in the clinical and experimental analysis. However, there are still unknown mechanisms for the cell cycle regulation, which is critical step for the progression of the cancer development. Especially it is not fully understood how the cells move to G1 phase from quiescent G0 phase in the mammalian cells. To find out the new gene networks associated with the two transition of the mammalian cell cycle (G0 to G1 and G1 to S phase), we analyzed the linkages between 39 representative oncogene or tumor suppressor genes, which related to the cell cycle regulation, with gene expression sets obtained from the publicly opened microarray data for mouse embryonic fibroblasts that synchronized by the serum starvation or hydroxyurea treatment. Analyses with a qualitative algorithm based on Bayesian networks that assume a log-linear relationship between genes have applied, and newly found networks were validated. Results highlighted the importance of two master genes, Cdk7 and Cdkna2 for the re-entry to G1 from G0, and suggested a new network connection from Cdk7 to downstream molecules, including the EGF receptor and N-myc. Introduction of a recombinant Cdk7 with retrovirus decreased endogenous EGFR and N-myc protein levels. The results supported the computational prediction of the Cdk7 network. Taken together, these result showed the existence of new regulating pathway from Cdk7 to Egfr and N-myc, suggesting this analytical approach provides an assessment of regulatory networks in complex mammalian cells, and the process of the carcinogenesis.

MycN is a transcriptional regulator of livin in neuroblastoma

Our previous studies have suggested that MycN may have a role in regulating livin expression in neuroblastoma. Here, we show that siRNA-mediated repression of MycN in neuroblastoma cells with both elevated MycN and livin expression resulted in significant downregulation of livin. Conversely, induction of MycN in neuroblastoma cells with low endogenous levels of MycN and livin protein upregulated livin expression. MycN directly associated with its regulatory motif (E-box) within the putative livin promoter. Based on these results, we hypothesize that MycN is required for livin expression and that livin may counteract the proapoptotic effects of MycN.

MYCN Promotes the Expansion of Phox2B-Positive Neuronal Progenitors to Drive Neuroblastoma Development

Amplification of the oncogene MYCN is a tumorigenic event in the development of a subset of neuroblastomas that commonly consist of undifferentiated or poorly differentiated neuroblasts with unfavorable clinical outcome. The cellular origin of these neuroblasts is unknown. Additionally, the cellular functions and target cells of MYCN in neuroblastoma development remain undefined. Here we examine the cell types that drive neuroblastoma development in TH-MYCN transgenic mice, an animal model of the human disease. Neuroblastoma development in these mice begins with hyperplastic lesions in early postnatal sympathetic ganglia. We show that both hyperplasia and primary tumors are composed predominantly of highly proliferative Phox2B(+) neuronal progenitors. MYCN induces the expansion of these progenitors by both promoting their proliferation and preventing their differentiation. We further identify a minor population of undifferentiated nestin(+) cells in both hyperplastic lesions and primary tumors that may serve as precursors of Phox2B(+) neuronal progenitors. These findings establish the identity of neuroblasts that characterize the tumor phenotype and suggest a cellular pathway by which MYCN can promote neuroblastoma development.

Stabilization of N-Myc Is a Critical Function of Aurora A in Human Neuroblastoma

In human neuroblastoma, amplification of the MYCN gene predicts poor prognosis and resistance to therapy. In a shRNA screen of genes that are highly expressed in MYCN-amplified tumors, we have identified AURKA as a gene that is required for the growth of MYCN-amplified neuroblastoma cells but largely dispensable for cells lacking amplified MYCN. Aurora A has a critical function in regulating turnover of the N-Myc protein. Degradation of N-Myc requires sequential phosphorylation by cyclin B/Cdk1 and Gsk3. N-Myc is therefore degraded during mitosis in response to low levels of PI3-kinase activity. Aurora A interacts with both N-Myc and the SCF(Fbxw7) ubiquitin ligase that ubiquitinates N-Myc and counteracts degradation of N-Myc, thereby uncoupling N-Myc stability from growth factor-dependent signals.

Cytotoxic activity of nemorosone in neuroblastoma cells

Neuroblastoma is the second most common solid tumour during childhood, characterized by rapid disease progression. Most children with metastasized neuroblastoma die despite intensive chemotherapy due to an intrinsic or acquired chemotherapy resistance. Thus, new therapeutic strategies are urgently needed. Here, we demonstrate that the novel compound nemorosone isolated from alcoholic extracts of Clusia rosea resins by reverse phase high pressure liquid chromatography (RP-HPLC) exerts cytotoxic activity in neuroblas-toma cell lines both parental and their clones selected for resistance against adriamycin, cisplatin, etoposide or 5-fluorouracil. Cell cycle studies revealed that nemorosone induces an accumulation in G0/G1- with a reduction in S-phase population combined with a robust up-regulation of p21Cip1. Furthermore, a dose-dependent apoptotic DNA laddering accompanied by an activation of caspase-3 activity was detected. Nemorosone induced a significant dephosphorylation of ERK1/2 in LAN-1 parental cells probably by the inhibition of its upstream kinase MEK1/2. No significant modulation of signal transducers JNK, p38 MAPK and Akt/PKB was detected. The enzymatic activity of immunoprecipitated Akt/PKB was strongly inhibited in vitro, suggesting that nemorosone exerts its anti-proliferative activity at least in part by targeting Akt/PKB in the cell lines studied. In addition, a synergistic effect with Raf-1 inhibitor BAY 43-9006 was found. Finally, nemorosone induced a considerable down-regulation of N-myc protein levels in parental LAN-1 and an etoposide resistant sub-line at the same drug-concentrations.

Distinct transcriptional MYCN/c-MYC activities are associated with spontaneous regression or malignant progression in neuroblastomas

Differences in MYCN/c-MYC target gene expression are associated with distinct neuroblastoma subtypes and clinical outcome.

High Skp2 Expression Characterizes High-Risk Neuroblastomas Independent of MYCN Status

Amplified MYCN oncogene defines a subgroup of neuroblastomas with poor outcome. However, a substantial number of MYCN single-copy neuroblastomas exhibits an aggressive phenotype similar to that of MYCN-amplified neuroblastomas even in the absence of high MYCN mRNA and/or protein levels. To identify shared molecular mechanisms that mediate the aggressive phenotype in MYCN-amplified and single-copy high-risk neuroblastomas, we defined genetic programs evoked by ectopically expressed MYCN in vitro and analyzed them in high-risk versus low-risk neuroblastoma tumors (n = 49) using cDNA microarrays. Candidate gene expression was validated in a separate cohort of 117 patients using quantitative PCR, and protein expression was analyzed in neuroblastoma tumors by immunoblotting and immunohistochemistry. We identified a genetic signature characterized by a subset of MYCN/MYC and E2F targets, including Skp2, encoding the F-box protein of the SCF(Skp2) E3-ligase, to be highly expressed in high-risk neuroblastomas independent of amplified MYCN. We validated the findings for Skp2 and analyzed its expression in relation to MYCN and E2F-1 expression in a separate cohort (n = 117) using quantitative PCR. High Skp2 expression proved to be a highly significant marker of dire prognosis independent of both MYCN status and disease stage, on the basis of multivariate analysis of event-free survival (hazard ratio, 3.54; 95% confidence interval, 1.56-8.00; P = 0.002). Skp2 protein expression was inversely correlated with expression of p27, the primary target of the SCF(Skp2) E3-ligase, in neuroblastoma tumors. Skp2 may have a key role in the progression of neuroblastomas and should make an attractive target for therapeutic approaches.

Inhibition of phosphatidylinositol 3-kinase destabilizes Mycn protein and blocks malignant progression in neuroblastoma.

Amplification of MYCN occurs commonly in neuroblastoma. We report that phosphatidylinositol 3-kinase (PI3K) inhibition in murine neuroblastoma (driven by a tyrosine hydroxylase-MYCN transgene) led to decreased tumor mass and decreased levels of Mycn protein without affecting levels of MYCN mRNA. Consistent with these observations, PI3K inhibition in MYCN-amplified human neuroblastoma cell lines resulted in decreased levels of Mycn protein without affecting levels of MYCN mRNA and caused decreased proliferation and increased apoptosis. To clarify the importance of Mycn as a target of broad-spectrum PI3K inhibitors, we transduced wild-type N-myc and N-myc mutants lacking glycogen synthase kinase 3beta phosphorylation sites into human neuroblastoma cells with no endogenous expression of myc. In contrast to wild-type N-myc, the phosphorylation-defective mutant proteins were stabilized and were resistant to the antiproliferative effects of PI3K inhibition. Our results show the importance of Mycn as a therapeutic target in established tumors in vivo, offer a mechanistic rationale to test PI3K inhibitors in MYCN-amplified neuroblastoma, and represent a therapeutic approach applicable to a broad range of cancers in which transcription factors are stabilized through a PI3K-dependent mechanism.

Cellular Retinoic Acid–Binding Protein II Is a Direct Transcriptional Target of MycN in Neuroblastoma

Neuroblastoma is a heterogeneous disease in which 22% of tumors show MycN oncogene amplification and are associated with poor clinical outcome. MycN is a transcription factor that regulates the expression of a number of proteins that affect the clinical behavior of neuroblastoma. We report here that cellular retinoic acid-binding protein II (CRABP-II) is a novel MycN target, expressed at significantly higher levels in primary neuroblastoma tumors with mycN oncogene amplification as compared with non-MycN-amplified tumors. Moreover, regulated induction and repression of MycN in a neuroblastoma-derived cell line resulted in temporal and proportionate expression of CRABP-II. CRABP-II is expressed in several cancers, but its role in tumorigenesis has not been elucidated. We show that MycN binds to the promoter of CRABP-II and induces CRABP-II transcription directly. In addition, CRABP-II-transfected neuroblastoma cell lines show an increase in MycN protein levels resulting in increased cell motility. Gene expression profiling of CRABP-II-expressing cell lines uncovered increased expression of the HuB (Hel N1) gene. Hu proteins have been implicated in regulating the stability of MycN mRNA and other mRNAs by binding to their 3' untranslated regions. We did not, however, observe any change in MycN mRNA stability or protein half-life in response to CRABP-II expression. In contrast, de novo MycN protein synthesis was increased in CRABP-II-expressing neuroblastoma cells, thereby suggesting an autoregulatory loop that might exacerbate the effects of MycN gene amplification and affect the clinical outcome. Our findings also suggest that CRABP-II may be a potential therapeutic target for neuroblastoma.

Key role for p27Kip1, retinoblastoma protein Rb, and MYCN in polyamine inhibitor-induced G1 cell cycle arrest in MYCN-amplified human neuroblastoma cells

Alpha-difluoromethylornithine (DFMO) inhibits the proto-oncogene ornithine decarboxylase (ODC) and is known to induce cell cycle arrest. However, the effect of DFMO on human neuroblastoma (NB) cells and the exact mechanism of DFMO-induced cell death are largely unknown. Treatment with DFMO in combination with SAM486A, an S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor, has been shown to enhance polyamine pool depletion. Therefore, we analysed the mechanism of action of DFMO and/or SAM486A in two established MYCN-amplified human NB cell lines. DFMO and SAM486A caused rapid cell growth inhibition, polyamine depletion, and G1 cell cycle arrest without apoptosis in cell lines LAN-1 and NMB-7. These effects were enhanced with combined inhibitors and largely prevented by cotreatment with exogenous polyamines. The G1 cell cycle arrest was concomitant with an increase in cyclin-dependent kinase inhibitor p27Kip1. In a similar fashion, DFMO and DFMO/SAM486A inhibited the phosphorylation of the G1/S transition-regulating retinoblastoma protein Rb at residues Ser795 and Ser807/811. Moreover, we observed a dramatic decrease in MYCN protein levels. Overexpression of MYCN induces an aggressive NB phenotype with malignant behavior. We show for the first time that DFMO and SAM486A induce G1 cell cycle arrest in NB cells through p27Kip1 and Rb hypophosphorylation.

N- MYC overexpression and neuroblastomas

In the advanced stages of human neuroblastomas, amplification of the N-MYC oncogene is a frequent event and correlates with poor prognosis and enhanced neovascularization. N-MYC amplification results in high N-MYC protein levels that can perturb the finely tuned interplay of N-MYC and MAX and could eventually induce abnormal expression patterns of target genes. However, to date few N-MYC target genes have been identified.Recently, Hatzi et al. questioned whether the N-MYC oncogene could stimulate tumor angiogenesis and thus enhance neuroblastoma progression [1xN-myc oncogene overexpression downregulates leukemia inhibitory factor in neuroblastoma. Hatzi, E. et al. Eur. J. Biochem. 2002; 269: 3732–3741Crossref | PubMed | Scopus (21)See all References[1]. Using a series of chromatographic steps, the authors isolated fractions from 47l of SH-EP007 supernatant that contained inhibitors of endothelial cell proliferation. One of the inhibitors identified was leukemia inhibitory factor (LIF). Its expression was dramatically downregulated in the presence of N-MYC overexpression, as determined using reporter assay experiments. This was significant because LIF has never before been reported to belong to a set of genes regulated by the MYC gene family members. The authors also found that downregulation of LIF was independent of N-MYC or MAX interaction and DNA binding because neither the HLH-Zip (helix-loop-helix zipper) nor the binding region deletion released the transcriptional repression. An intriguing observation is that the contiguous N-terminal N-MYC is required for repression because deletion of either the MbI (MYC box I) and MbII domains did not release the N-MYC mutants from the suppressive effect of the LIF promoter.In conclusion, N-MYC amplification could tilt the balance of angiomodulators in favor of the stimulators by downregulating LIF and thus participate in increasing neovascularization. Considering the functional similarities between C-MYC and N-MYC, data presented in this study correlate very well with results observed in mouse models. In these, the deregulated expression of C-MYC alone, in combination with suppressed apoptosis, is sufficient to give reversible angiogenic tumors, without the apparent need for a sudden ‘angiogenic switch’.

Role of N-myc in the Developing Mouse Kidney

N-myc is a transcription factor expressed in the developing metanephric kidney and other organs. In mice, complete disruption of the N-myc gene results in fetal death on the first day of renal organogenesis. In addition to the null N-myc allele, others have generated a hypomorphic N-myc allele. In this study, combinations of these mutant genes were used to demonstrate that reduction in N-myc protein levels correlate with fewer developing glomeruli and collecting ducts in embryonic kidney explants. Histological sections revealed that the mutant kidneys were hypoplastic with normal developing structures. The data indicate that the hypoplasia is due to a reduction in proliferation rather than an increase in apoptosis. Thus, N-myc loss causes a decrease in numbers of ureteric bud tips and developing glomeruli in explants and hypoplastic kidneys in vivo, in a dose-dependent manner.

Induction of Neuronal Differentiation by p73 in a Neuroblastoma Cell Line

The p53-related p73 and p63 genes encode proteins that share considerable structural and functional homology with p53. Despite similarities, their deletion in mice has different outcomes, implying that the three genes may play distinct roles in vivo. Here we show that endogenous p73 levels increase in neuroblastoma cells induced to differentiate by retinoic acid and that exogenously expressed p73, but not p53, is sufficient to induce both morphological (neurite outgrowth) and biochemical (expression of neurofilaments and neural cell adhesion molecule (N-CAM); down-regulation of N-MYC and up-regulation of pRB) markers of neuronal differentiation. This activity is shared, to different extents, by all p73 isoforms, whereas the transcriptionally inactive mutants of p73 isoforms are ineffective. Conversely, blockage of endogenous p73 isoforms with a dominant negative p73 results in the abrogation of retinoid-induced N-CAM promoter-driven transcription. Our results indicate that the p73 isoforms activate a pathway that is not shared by p53 and that is required for neuroblastoma cell differentiation in vitro.