MYCN Research Articles

Abstract 3510: N-Myc positively influences mitochondrial biogenesis in neuroblastoma

Abstract Background: Neuroblastoma is the most common extracranial tumor of childhood., accounting for approximately 7% of all cancer in children. The morbidity and mortality are affected by a number of factors - perhaps most significantly by amplification of the oncogene, N-MYC. Advanced stage disease with N-MYC overexpression confers the highest mortality. The N-MYC gene product (N-Myc) regulates the transcription of many genes, and when overexpressed is thought to enhance the cell cycle and dysregulate apoptosis. The function of N-Myc is virtually identical to another oncoprotein of the same family, c-Myc, yet is restricted by developmental stage and cell type. Overexpression of c-Myc has been shown to increase mitochondrial biogenesis by alterations in mitochondrial dynamics. It has been proposed that these changes may contribute to c-Myc induced tumorigenesis. We hypothesize that N-Myc amplification will have similar effects on mitochondrial biogenesis in neuroblastoma. Design/Methods: To perform these studies we utilized a cell line with endogenous levels of N-Myc (SK-N-SH). In these cells we have ectopically overexpressed N-Myc and examined the cells for changes in the mitochondria with various assays, including: Western blots, electron microscopy, flow cytometry, ATP assay and confocal microscopy. Results: We found a X-fold increase in mitochondrial mass in cells that overexpress N-Myc,, as well as a more tubular appearance with less organized cristae. Additionally, N-Myc changed the expression of various mitochondrial fusion and fission proteins. The most dramatic change was seen in the expression of the fusion protein Opa1, which changed by ten-fold. This, combined with a decrease in expression of fission proteins DRP1 and MFF, may account for the more tubular appearance of mitochondria in N-Myc overexpressing cells. Conclusions N-Myc amplification appeared to increase mitochondrial mass. This may occur as a result of increased expression of mitochondrial fusion proteins, as previously observed in c-Myc overexpressing cells. Additionally, mitochondrial function is influenced by N-Myc overexpression. Therefore, N-Myc amplification appears to positively influence mitochondrial biogenesis in neuroblastoma. Further studies are needed to determine if this increase in mitochondrial mass and function is necessary for the development and maintenance of neuroblastoma. Citation Format: Manika Sharma, James A. Graves. N-Myc positively influences mitochondrial biogenesis in neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3510. doi:10.1158/1538-7445.AM2014-3510

Abstract LB-68: MYCN-induced TRPM7 expression and channel activity occurs through a mechanism that involves ornithine decarboxylase

Abstract Neuroblastoma (NB) is an extra-cranial solid cancer in children. MYCN is a transcription factor that regulates the expression of genes that are involved in cell proliferation, growth, cell migration, etc. MYCN gene amplification is a prognostic indicator of poor outcome in NB. Recent studies have shown that intracellular free calcium regulates multiple steps involved in cell migration. Although significant advances have been made in understanding the role of calcium during migration, little has been achieved towards understanding its impact on the malignant progression of NB. Our results showed that MYCN gene amplification or over-expression of MYCN in NB cells increased TRPM7 expression, increased peak TRPM7 current, induced constitutive TRPM7 channel activity, enhanced calcium signaling, and promoted cell migration, implicating TRPM7 as a potential regulator of calcium signals that promote NB cell migration and invasion. Recent results suggest that MYCN may regulate TRPM7 expression through a process that involves ornithine decarboxylase (ODC), a rate limiting enzyme in the biosynthesis of polyamines, and a direct transcriptional target of MYCN. Treatment of NB cells with α-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, or transfection of ODC specific siRNA effectively decreased TRPM7 expression. This effect was attenuated by supplementing the culture media with spermidine, confirming that ODC may function downstream of MYCN to regulate TRPM7 expression. Interestingly, western blot analysis of NB cell lysates showed that MYCN induced the cleavage of the TRPM7 kinase domain, a post-translational modification that has been shown to increase TRPM7 currents and induce constitutive TRPM7 channel activity. Additionally, DFMO inhibited TRPM7 cleavage, an effect that was reversed by spermidine supplementation. Taken together, these results suggest that MYCN may regulate TRPM7 expression and channel activity through a mechanism that involves ODC-mediated polyamine biosynthesis. Citation Format: Dana-Lynn T. Koomoa, Ingo Lange. MYCN-induced TRPM7 expression and channel activity occurs through a mechanism that involves ornithine decarboxylase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-68. doi:10.1158/1538-7445.AM2014-LB-68

Abstract LB-125: Selective inhibition of CDK7 targets MYCN-driven transcriptional amplification in neuroblastoma

Abstract Oncogenic MYC family transcription factors act as universal amplifiers of the existing gene expression program in many cancer cells, thus reducing rate-limiting constraints on growth and proliferation. Amplification of the MYCN gene defines approximately 50% of high risk neuroblastomas (NB), and is associated with aggressive disease and a poor clinical outcome. Here we exploit MYCN-driven global transcriptional amplification to specifically target MYCN-deregulated NB cells by inhibiting CDK7, a cyclin-dependent kinase with major roles in transcriptional initiation (as part of the TFIIH complex) and elongation (by activating CDK9/P-TEFb). For this purpose, we chose CDK7-IN-1, a newly developed, highly selective, first-in-class covalent inhibitor of CDK7, and then determined the effects of CDK7 inhibition on MYCN expression and global transcriptional activity. NB cells expressing high levels of MYCN were 10 times more sensitive to CDK7 inhibition than normal cells or NB cells not driven by amplified MYCN. CDK7-IN-1 was more active than its reversible (non-covalent) analogue and two pan-CDK inhibitors, roscovitine and flavopiridol. Cytotoxicity in treated MYCN-amplified NB cells resulted from G2 arrest and apoptosis. We observed a dose-dependent decrease in serine 2, 5 and 7 phosphorylation of RNA Pol II C-terminal domain only in MYCN-amplified NB cells, indicating that CDK7-IN-1 selectively inhibits RNA Pol II-mediated transcriptional initiation and elongation. Growth inhibition was accompanied by downregulation of MYCN and MYCN-associated transcriptional programs. CDK7-IN-1 significantly slowed tumor growth in a xenograft model of MYCN-amplified NB (median growth, 56.8% vs. 100% for vehicle-treated mice, P <0.05; n=6 per group) with tumors showing decreased MYCN expression. Mice remained free of toxicity over 4 weeks of CDK7-IN-1 treatment, suggesting that a therapeutic window may exist for NB cells with high MYCN expression. In conclusion, we show for the first time that selective suppression of MYCN expression and MYCN-associated transcriptional activity can be achieved through CDK7 inhibition, with associated antitumor effects in high-risk NB. Thus, CDK7 inhibition warrants further attention as a potential therapeutic strategy for MYCN-deregulated NB and perhaps other MYC-driven cancers. Citation Format: Edmond Chipumuro, Eugenio Marco, Tinghu Zhang, Camilla Christensen, Nicholas Kwiatkowski, Bandana Sharma, Clark Hatheway, Abigail Altabef, Brian J. Abraham, Kwok-Kin Wong, Guo-Cheng Yuan, Richard A. Young, Nathanael S. Gray, Rani E. George. Selective inhibition of CDK7 targets MYCN-driven transcriptional amplification in neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-125. doi:10.1158/1538-7445.AM2014-LB-125

Abstract 1900: Prognostic significance of genetic aberrations in neuroblastoma

Abstract Clinical behavior of neuroblastoma varies due to differences in biological characteristics of the tumor. 1p, 11q deletions, MYCN amplification (MNA) are known to be adverse prognostic markers while significance of many other copy number variations (CNV) is less clear. We analyzed 108 primary tumors with SALSA P251 and P252 MLPA neuroblastoma kits specific for loci 1p,2p,3p,11q,17q and 100 with P253 kit (4p,7q,9p,12q,14q). Prognostic significance was estimated by event-free survival (EFS) with median of follow-up time 28 months (range 1-166 months). MLPA revealed MNA in 19 patients (17.6%), in 12 cases (11.1%) NAG, DDX1 or ALK genes were coamplified with MYCN. EFS in case of MNA was significantly low comparing with normal MYCN status (0.20±0.11 vs. 0.69±0.06, p<0.001). 2p23-24 gain was observed in 15 patients (13.9%) and showed prognostic significance in patients under 1 year (EFS 0.53±0.25 vs. 0.96±0.04, p=0.047). In 29 patients (26.9%) 1p deletion was revealed. It had prognostic impact in the whole group (EFS 0.33±0.10 vs. 0.67±0.06, p=0.002) and in MYCN non-amplified patients (EFS 0.40±0.14 vs. 0.71±0.06, p=0.029). 17q gain was detected in 54 patients (50.0%) and led to decreased EFS in all patients (0.42±0.08 vs. 0.71±0.07, p<0.001) as well as in MYCN-negative subgroup (0.47±0.10 vs. 0.77±0.07, p=0.002). 4p gain detected in 8 patients (8.0%) dramatically decreased EFS in patients under 1 year of age (0.00 vs. 0.88±0.06, p=0.055). Gain of short and long arms of chromosome 7 detected in 6 patients (6.0%) led to reduced EFS in the whole group (0.33±0.19 vs. 0.56±0.06, p=0.053) and in MYCN non-amplified patients: 0.40±0.22 vs. 0.64±0.06, p=0.044. In 8 patients (8.0%) 9p deletion was found. Presence of this aberration resulted in vivid decreasing of EFS in both all evaluated patients (0.00 vs. 0.60±0.06, p=0.035) and MYCN non-amplified group (0.00 vs. 0.68±0.06, p=0.047). 3p deletion observed in 30 of 108 patients (27.8%) had adverse prognostic significance in the group of patients above 3 years of age (EFS 0.25±0.23 vs. 0.71±0.11, p=0.034). Neither interstitial (n=16, 14.8%) nor terminal (n=9, 8.3%) 11q deletions had prognostic significance in our series: EFS 0.28±0.14 and 0.78±0.14 vs. 0.60±0.06, p=0.164 and p=0.477 respectively. No influence on EFS was found in case of presence 12q or MDM2 gene gain (n= 20, 20.0%) and 14q aberrations (n=14, 14.0%). Thus, MLPA is accurate technique for CNV detection in neuroblastoma. In our cohort of patients, treated according GPOH NB2004 protocol, MNA, 1p, 9p deletions and 17q gain demonstrated negative influence on EFS. Presence of 2p23-24 gain and 4p gain led to decrease EFS in the group of patients below 1 year old, while 1p and 9p deletions, 17q gain and gain of both arms of chromosome 7 retained prognostic significance in MYCN non-amplified patients. Coamplification of MYCN gene with DDX1, NAG and ALK genes had no prognostic significance comparing with amplification of MYCN gene alone. Citation Format: Alexander E. Druy, Grigory A. Tsaur, Egor V. Shorikov, Alexander M. Popov, Leonid I. Saveliev, Larisa G. Fechina. Prognostic significance of genetic aberrations in neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1900. doi:10.1158/1538-7445.AM2014-1900

Multidisciplinary management of cervical neuroblastoma in infants

ObjectivesNeuroblastoma is the most common malignancy in infancy, it is a histologically and genetically heterogeneous tumor, the therapy and outcome of which is influenced by age, histological variant and genetic background as well. MethodsWe present two consecutive infant patients with neuroblastoma of the neck discussing the etiology, the diagnosis and the surgical and oncological treatment of the tumor, which was observed in a relatively rare manifestation in the head–neck region. ResultsOur first patient (age: 5.5 months) was MYCN (v-myc myelocytomatosis viral related oncogene, neuroblastoma derived) negative, INSS (International Neuroblastoma Staging System) Stage 3 and INRGSS (International Neuroblastoma Risk Group Staging System) Stage 3 because of the contralateral lymph node involvement while the complete gross resection of the primary tumor mass was feasible. The patient is tumor free after three years of follow-up. Our second patient (age: 5 months) was MYCN negative, INSS Stage 2 and INRGSS Stage 1, as both the primary tumor and the ipsilateral lymph nodes were totally removed via a modified radical neck dissection. The patient is tumor free after three years of follow-up. ConclusionFor MYCN negative patients, especially in early age, the prognosis of neuroblastoma is good, surgical resection and chemotherapy together is an adequate treatment protocol (as in our two patients). While MYCN-amplified patients require a combined and aggressive treatment with surgery, chemotherapy, radiotherapy, and immunotherapy to be able to obtain a favorable survival rate according to the literature.

Construction and identification of the lentivirus vector targeting MYCN gene of human neuroblastoma cells

Objective To construct the lentivirus vector targeting MYCN of human neuroblastoma cells and identify its silencing effects on MYCN gene.Methods Four interference plasmids targeting MYCN was designed and constructed based on human MYCN gene information.After screening and identification,they were restructured with the pLenti6.3/V5 Dest vector,and packed with the lentivims to construct the Lenti-EGFP-MYCN-miR lentiviral vector,which then infected the SK-N-BE (2) cells.The silencing effects of these four Lenti-EGFP-MYCN-miR lentiviral vectors on the MYCN mRNA and protein expressions were detected using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting,meanwhile,the lentiviral vector with the best interference target sequence was screened.Results Digestion and sequencing confirmed that four MYCN-targeting Lenti-EGFP-MYCN-miR lentiviral vectors were successfully constructed.These four lentiviral vectors were infected into the SK-N-BE (2) cells.As shown by FQ-PCR and Western blotting,the mRNA and protein expression levels of MYCN were 0.577±0.015,0.333 ±0.016,0.090 ±0.010,0.197 ±0.015 and 0.833 ±0.049,0.563 ±0.040,0.300 ± 0.020,0.387 ± 0.015,respectively,among which the Lenti-EGFP-MYCN-3-miR showed most potent inhibitory effect on MYCN and therefore was selected for the the best RNAi target sequence.Condusion The successfully constructed Lenti-EGFP-MYCN-miR lentivirus vector targeting MYCN could effectively infect SK-N-BE (2) cells in vitro and significantly inhibit mRNA and protein expression of MYCN. Key words: Neuroblastomas ; MYCN ; RNA interfere ; Lentiviral vector

A PCNA-derived cell permeable peptide selectively inhibits neuroblastoma cell growth.

Proliferating cell nuclear antigen (PCNA), through its interaction with various proteins involved in DNA synthesis, cell cycle regulation, and DNA repair, plays a central role in maintaining genome stability. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was significantly expressed in a broad range of cancer cells and tumor tissues, but not in non-malignant cells. We found that the caPCNA-specific antigenic site lies between L126 and Y133, a region within the interconnector domain of PCNA that is known to be a major binding site for many of PCNA's interacting proteins. We hypothesized that therapeutic agents targeting protein-protein interactions mediated through this region may confer differential toxicity to normal and malignant cells. To test this hypothesis, we designed a cell permeable peptide containing the PCNA L126-Y133 sequence. Here, we report that this peptide selectively kills human neuroblastoma cells, especially those with MYCN gene amplification, with much less toxicity to non-malignant human cells. Mechanistically, the peptide is able to block PCNA interactions in cancer cells. It interferes with DNA synthesis and homologous recombination-mediated double-stranded DNA break repair, resulting in S-phase arrest, accumulation of DNA damage, and enhanced sensitivity to cisplatin. These results demonstrate conceptually the utility of this peptide for treating neuroblastomas, particularly, the unfavorable MYCN-amplified tumors.

Metachronous Neuroblastoma in an Infant With Germline Translocation Resulting in Partial Trisomy 2p

A male infant with dysmorphic features, intestinal malrotation, and developmental delay was found to have a germline translocation resulting in partial trisomy 2p and monosomy 16p. At 3 and 9 months of age, he developed localized neuroblastoma in each adrenal, which was managed with surgical resection. Tumors were MYCN non-amplified, with 2p copy gain consistent with the germline translocation. The potential increased risk of neuroblastoma associated with partial trisomy 2p is discussed in the context of this and previously published cases, and may be due to increased constitutional expression of MYCN and ALK genes, both located within the duplicated 2p region.

Aryl Hydrocarbon Receptor Downregulates MYCN Expression and Promotes Cell Differentiation of Neuroblastoma

Neuroblastoma (NB) is the most common malignant disease of infancy. MYCN amplification is a prognostic factor for NB and is a sign of highly malignant disease and poor patient prognosis. In this study, we aimed to investigate novel MYCN-related genes and assess how they affect NB cell behavior. The different gene expression found in 10 MYCN amplification NB tumors and 10 tumors with normal MYCN copy number were analyzed using tissue oligonucleotide microarrays. Ingenuity Pathway Analysis was subsequently performed to identify the potential genes involved in MYCN regulation pathways. Aryl hydrocarbon receptor (AHR), a receptor for dioxin-like compounds, was found to be inversely correlated with MYCN expression in NB tissues. This correlation was confirmed in a further 14 human NB samples. Moreover, AHR expression in NB tumors was found to correlate highly with histological grade of differentiation. In vitro studies revealed that AHR overexpression in NB cells induced spontaneous cell differentiation. In addition, it was found that ectopic expression of AHR suppressed MYCN promoter activity resulting in downregulation of MYCN expression. The suppression effect of AHR on the transcription of MYCN was compensated for by E2F1 overexpression, indicating that E2F1 is involved in the AHR-regulating MYCN pathway. Furthermore, AHR shRNA promotes the expression of E2F1 and MYCN in NB cells. These findings suggest that AHR is one of the upstream regulators of MYCN. Through the modulation of E2F1, AHR regulates MYCN gene expression, which may in turn affect NB differentiation.

A Cell-Based High-Throughput Screen Addressing 3′UTR-Dependent Regulation of the MYCN Gene

Neuroblastoma is the most common extracranial solid tumor of infancy. Amplification of MYCN oncogene is found in approximately 20 % of all neuroblastoma patients and correlates with advanced disease stages, rapid tumor progression, and poor prognosis, making this gene an obvious therapeutic target. However, being a transcriptional factor MYCN is difficult for pharmacological targeting, and there are currently no clinical trials aiming MYCN protein directly. Here we describe an alternative approach to address deregulated MYCN expression. In particular, we focus on the role of a 3′ untranslated region (3′UTR) of the MYCN gene in the modulation of its mRNA fate and identification of compounds able to affect it. The luciferase reporter construct with the full length MYCN 3′UTR was generated and subsequently integrated in the CHP134 neuroblastoma cell line. After validation, the assay was used to screen a 2000 compound library. Molecules affecting luciferase activity were checked for reproducibility and counter-screened for promoter effects and cytotoxic activity resulting in selection of four hits. We propose this cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms. Identification of such compounds could potentially result in development of clinically relevant therapeutics for various diseases including neuroblastoma.Electronic supplementary materialThe online version of this article (doi:10.1007/s12033-014-9739-z) contains supplementary material, which is available to authorized users.

Inactivation of SMC2 shows a synergistic lethal response in MYCN-amplified neuroblastoma cells

The condensin complex is required for chromosome condensation during mitosis; however, the role of this complex during interphase is unclear. Neuroblastoma is the most common extracranial solid tumor of childhood, and it is often lethal. In human neuroblastoma, MYCN gene amplification is correlated with poor prognosis. This study demonstrates that the gene encoding the condensin complex subunit SMC2 is transcriptionally regulated by MYCN. SMC2 also transcriptionally regulates DNA damage response genes in cooperation with MYCN. Downregulation of SMC2 induced DNA damage and showed a synergistic lethal response in MYCN-amplified/overexpression cells, leading to apoptosis in human neuroblastoma cells. Finally, this study found that patients bearing MYCN-amplified tumors showed improved survival when SMC2 expression was low. These results identify novel functions of SMC2 in DNA damage response, and we propose that SMC2 (or the condensin complex) is a novel molecular target for the treatment of MYCN-amplified neuroblastoma.

MYCN gene expression is required for the onset of the differentiation programme in neuroblastoma cells

Neuroblastoma is an embryonic tumour of the sympathetic nervous system and is one of the most common cancers in childhood. A high differentiation stage has been associated with a favourable outcome; however, the mechanisms governing neuroblastoma cell differentiation are not completely understood. The MYCN gene is considered the hallmark of neuroblastoma. Even though it has been reported that MYCN has a role during embryonic development, it is needed its decrease so that differentiation can be completed. We aimed to better define the role of MYCN in the differentiation processes, particularly during the early stages. Considering the ability of MYCN to regulate non-coding RNAs, our hypothesis was that N-Myc protein might be necessary to activate differentiation (mimicking embryonic development events) by regulating miRNAs critical for this process. We show that MYCN expression increased in embryonic cortical neural precursor cells at an early stage after differentiation induction. To investigate our hypothesis, we used human neuroblastoma cell lines. In LAN-5 neuroblastoma cells, MYCN was upregulated after 2 days of differentiation induction before its expected downregulation. Positive modulation of various differentiation markers was associated with the increased MYCN expression. Similarly, MYCN silencing inhibited such differentiation, leading to negative modulation of various differentiation markers. Furthermore, MYCN gene overexpression in the poorly differentiating neuroblastoma cell line SK-N-AS restored the ability of such cells to differentiate. We identified three key miRNAs, which could regulate the onset of differentiation programme in the neuroblastoma cells in which we modulated MYCN. Interestingly, these effects were accompanied by changes in the apoptotic compartment evaluated both as expression of apoptosis-related genes and as fraction of apoptotic cells. Therefore, our idea is that MYCN is necessary during the activation of neuroblastoma differentiation to induce apoptosis in cells that are not committed to differentiate.

RB1 gene inactivation by chromothripsis in human retinoblastoma

Retinoblastoma is a rare childhood cancer of the developing retina. Most retinoblastomas initiate with biallelic inactivation of the RB1 gene through diverse mechanisms including point mutations, nucleotide insertions, deletions, loss of heterozygosity and promoter hypermethylation. Recently, a novel mechanism of retinoblastoma initiation was proposed. Gallie and colleagues discovered that a small proportion of retinoblastomas lack RB1 mutations and had MYCN amplification [1]. In this study, we identified recurrent chromosomal, regional and focal genomic lesions in 94 primary retinoblastomas with their matched normal DNA using SNP 6.0 chips. We also analyzed the RB1 gene mutations and compared the mechanism of RB1 inactivation to the recurrent copy number variations in the retinoblastoma genome. In addition to the previously described focal amplification of MYCN and deletions in RB1 and BCOR, we also identified recurrent focal amplification of OTX2, a transcription factor required for retinal photoreceptor development. We identified 10 retinoblastomas in our cohort that lacked RB1 point mutations or indels. We performed whole genome sequencing on those 10 tumors and their corresponding germline DNA. In one of the tumors, the RB1 gene was unaltered, the MYCN gene was amplified and RB1 protein was expressed in the nuclei of the tumor cells. In addition, several tumors had complex patterns of structural variations and we identified 3 tumors with chromothripsis at the RB1 locus. This is the first report of chromothripsis as a mechanism for RB1 gene inactivation in cancer.

MycN promotes TRPM7 expression and cell migration in neuroblastoma through a process that involves polyamines

Neuroblastoma is an extra-cranial solid cancer in children. MYCN gene amplification is a prognostic indicator of poor outcome in neuroblastoma. Recent studies have shown that the multiple steps involved in cell migration are dependent on the availability of intracellular calcium (Ca2+). Although significant advances have been made in understanding the role of Ca2+ during migration, little has been achieved towards understanding its impact on the progression of diseases such as cancer. Interestingly, previous studies showed that cancer cell migration is regulated by TRPM7, a calcium-permeable ion channel. The objective of the current study was to elucidate the mechanism by which MycN promotes NB cell migration and the mechanism regulating TRPM7 expression. The results showed that MycN increased TRPM7 expression, induced TRPM7 channel activity, increased intracellular Ca2+ signaling, and promoted cell migration in NB cells. The results also showed that inhibition or down-regulation of ornithine decarboxylase (ODC) inhibited TRPM7 expression, a process that was reversed by spermidine. Overall, this study provides evidence that MycN promotes TRPM7 expression and cell migration through a mechanism that involves ODC synthesis of polyamines.

MYC FAMILY GENES IN SOLID-PSEUDOPAPILLARY TUMORS OF THE PANCREAS

In the paper the study of molecular disorder genes c-Myc and N-Myc in solid-pseudopapillary tumor of the pancreas. Examined 24 samples of tumors obtained from 21 patients, including 19 primary, recurrent 1, 4 metastasis. Fluorescence in situ hybridization was performed using DNA samples lokusspetsificheskih to the loci 2p24, 6q23, 8q24 company «Vysis, Abbott Laboratories Inc», demonstrated that amplification of the N-Myc gene in solid-pseudopapillary tumor can be another confirming its neurogenic origin.

Involvement of germline DDX1–MYCN duplication in inherited nephroblastoma

This report concerns a 3-year-old girl with prenatal bilateral nephroblastomatosis and a family history of nephroblastoma. This girl had a chromosome 8 pericentric inversion inherited from her father. This inversion was observed in healthy individuals of the family and was absent in other individuals suffering from embryonic kidney tumor. We then supposed that another genetic anomaly predisposed her to tumorogenesis. Additional cryptic imbalances are reported in cases of apparently balanced chromosomal rearrangements with an abnormal phenotype. Array-CGH analysis showed a 569 kb duplication at 2p24.3 including the DDX1 and MYCN genes. This duplication was inherited from the patient's father who also had a nephroblastoma. A link between germline MYCN duplication and the occurrence of other embryonic cancers such as neuroblastoma has already been described. We supposed that germline DDX1–MYCN duplication could also be involved in the apparition of nephroblastomas.

Detection of MYCN amplification using blood plasma: noninvasive therapy evaluation and prediction of prognosis in neuroblastoma

Amplification of neuroblastoma derived (avian)v-myc myelocytomatosis viral related oncogene (MYCN) is an important risk-stratified indicator in neuroblastoma. To evaluate the feasibility of noninvasive measurement of MYCN amplification, we analyzed MYCN amplification in stored blood plasma samples. We used quantitative real-time PCR to determine MYCN copy numbers in plasma-derived DNA of 10 healthy volunteers and 50 neuroblastoma cases. The copy number was calculated as the ratio of copies of MYCN to those of a reference gene. Plasma samples obtained after surgery or neoadjuvant therapy were also analyzed in five cases and four cases, respectively. In 34 neuroblastoma cases, MYCN was nonamplified in both tumor tissue and blood plasma. In 16 neuroblastoma cases, MYCN was amplified in both tumor tissue and blood plasma; 13 of the 16 cases showed poor outcomes. MYCN amplification was undetectable in blood plasma shortly after surgery or neoadjuvant therapy. The correlation coefficient between MYCN copy numbers in tumor tissue and in blood plasma was approximately 0.9. We can detect MYCN amplification of tumor tissue noninvasively and quantitatively by measuring the MYCN copy number in blood plasma. Determination of MYCN copy number in plasma may be useful when evaluating surgery and neoadjuvant chemotherapy.

Solution equilibria of cytosine- and guanine-rich sequences near the promoter region of the n-myc gene that contain stable hairpins within lateral loops

BackgroundCytosine- and guanine-rich regions of DNA are capable of forming complex structures named i-motifs and G-quadruplexes, respectively. In the present study the solution equilibria at nearly physiological conditions of a 34-base long cytosine-rich sequence and its complementary guanine-rich strand corresponding to the first intron of the n-myc gene were studied. Both sequences, not yet studied, contain a 12-base tract capable of forming stable hairpins inside the i-motif and G-quadruplex structures, respectively. MethodsSpectroscopic, mass spectrometry and separation techniques, as well as multivariate data analysis methods, were used to unravel the species and conformations present. ResultsThe cytosine-rich sequence forms two i-motifs that differ in the protonation of bases located in the loops. A stable Watson–Crick hairpin is formed by the bases in the first loop, stabilizing the i-motif structure. The guanine-rich sequence adopts a parallel G-quadruplex structure that is stable throughout the pH range 3–7, despite the protonation of cytosine and adenine bases at lower pH values. The presence of G-quadruplex aggregates was confirmed using separation techniques. When mixed, G-quadruplex and i-motif coexist with the Watson–Crick duplex across a pH range from approximately 3.0 to 6.5. ConclusionsTwo cytosine- and guanine-rich sequences in n-myc gene may form stable i-motif and G-quadruplex structures even in the presence of long loops. pH modulates the equilibria involving the intramolecular structures and the intermolecular Watson–Crick duplex. General significanceWatson–Crick hairpins located in the intramolecular G-quadruplexes and i-motifs in the promoter regions of oncogenes could play a role in stabilizing these structures.

Abstract A60: Analysis of gene copy number changes in chronic lymphocytic leukemia

Abstract Chronic lymphocytic leukemia (CLL) is the most frequent type of leukemia in the elderly and is accompanied by the accumulation of malignant monoclonal B-cells in the bone marrow, peripheral blood or lymph nodes which harbor various genetic aberrations. Genomic instability is an important characteristic of leukemia cells and plays a role both in its development as well as in the response to therapy. Somatic copy number changes in distinct genes are associated with the development of specific leukemias. Copy number variations are important diagnostic tools and help in classifying the disease and guiding the therapy. Current strategies for the detection of chromosomal aberrations in CLL are laborious and time consuming. Using multiplex ligation dependent probe amplification (MLPA) copy number changes at multiple loci can be detected simultaneously in a single simple PCR reaction. In this study, we investigated the copy number alterations in several key genes in CLL by MLPA. DNA was isolated from the venous blood samples from 92 patients with CLL and 10 healthy controls. Copy number changes of 20 genes were analyzed at 29 different sites by MLPA. DNA samples were hybridized with the sequence specific probes for each gene and the hybridized probe pairs were ligated. PCR reaction was performed to amplify the ligated probes. PCR products were separated by capillary electrophoresis using the ABI Prism 310 genetic analyzer. The peaks were evaluated for the genomic gains and losses after normalization of each peak. Reference genes were used for normalization of each gene signal. The data from the patients were compared with control samples from healthy individuals. Various genetic aberrations were detected in the patients with CLL. The most frequently observed genetic aberrations were losses in the chromosomal regions 13q14.3 and 13q14.2 and involved the DLEU7 (49 %), DLEU2 (41 %), KCNRG (35.9 %) and RB1 (14 %) genes. Copy number losses in the MYCN, ESR1, EIF3S3, CDKN2A, CDKN2B, ATM, CHFR and TP53 genes were lower with a frequency ranging between 1-10 %. The most frequently amplified gene was the MYC gene (30.4 %), followed by the LRMP (17.4 %), ESR1 (15 %) and CHFR (14 %) genes. Gains in the MYCN, IGF2R, PARK2, CCND2, CDK4, IFNG and LDLR genes were observed with a lower frequency. Our data indicate that copy number changes in several important genes are frequent in the pathogenesis of CLL. Simultaneous analysis and characterization of these genetic alterations can be performed using MLPA and the test can provide valuable clinical information when employed as a first-line screening approach for CLL patients. Citation Format: Nejat Dalay, Mustafa Isin, Guven Cetin, Melih Aktan. Analysis of gene copy number changes in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A60.

Target delivery of MYCN siRNA by folate-nanoliposomes delivery system in a metastatic neuroblastoma model

BackgroundFolate-nanoliposomes delivery system has emerged recently as a specific and safety delivery method and gradually used as the carrier of a variety kinds of drugs including compounds, plasmids and siRNAs.MethodsIn this study, we established a bone marrow and bone metastasis xenograft mouse model by injecting the LA-N-5 cell into the bone marrow cavity. Fluorescence microscopy, TUNEL Assay, Quantitative RT-PCR and western blot were conducted to analysis the distribution of folate-nanoliposomes entrapped MYCN (V-myc myelocytomatosis viral related oncogene) siRNA in mice and the relevant suppression effect.ResultsThe folate-nanoliposomes entrapped MYCN siRNA can be specifically distributed in tumor tissues. Further study shows that folate-nanoliposomes entrapped MYCN siRNA lead to MYCN mRNA expression significantly down-regulated (>50%, and p < 0.05) compared with negative control siRNA treatment. MYCN protein expression was inhibited about 60% in vivo, thus induced tumor cell apoptosis markedly.ConclusionThis study point to a new way for treatment of metastatic neuroblastoma and could widen the application of folate-nanoliposomes delivery system in tumor therapy.