Complexity of N-Myc transcriptional function in childhood neuroblastoma

Myc is a transcription factor that can activate transcription of several hundreds genes by direct binding to their promoters at specific DNA sequences (E-box). However, recent studies have also shown that it can exert its biological role by repressing transcription. Such studies collectively support a model in which c-Myc-mediated repression occurs through interactions with transcription factors bound to promoter DNA regions but not through direct recognition of typical E-box sequences. Here, we investigated whether N-Myc can also repress gene transcription, and how this is mechanistically achieved. We used human neuroblastoma cells as a model system in that N-MYC amplification/over-expression represents a key prognostic marker of this tumour. By means of transcription profile analyses we could identify at least 5 genes (TRKA, p75NTR, ABCC3, TG2, p21) that are specifically repressed by N-Myc. Through a dual-step-ChIP assay and genetic dissection of gene promoters, we found that N-Myc is physically associated with gene promoters in vivo, in proximity of the transcription start site. N-Myc association with promoters requires interaction with other proteins, such as Sp1 and Miz1 transcription factors. Furthermore, we found that N-Myc may repress gene expression by interfering directly with Sp1 and/or with Miz1 activity (i.e. TRKA, p75NTR, ABCC3, p21) or by recruiting Histone Deacetylase 1 (Hdac1) (i.e. TG2). In vitro analyses show that distinct N-Myc domains can interact with Sp1, Miz1 and Hdac1, supporting the idea that Myc may participate in distinct repression complexes by interacting specifically with diverse proteins. Finally, results show that N-Myc, through repressed genes, affects important cellular functions, such as apoptosis, growth, differentiation and motility. Overall, our results support a model in which N-Myc, like c-Myc, can repress gene transcription by direct interaction with Sp1 and/or Miz1, and provide further lines of evidence on the importance of transcriptional repression by Myc factors in tumour biology.

Preclinical neuroblastoma models for a pharmacological study of a new MYCN oncogene inhibitor

Background. Neuroblastoma is the most deadly solid tumor of childhood. In the 25% of cases it is associated with MYCN amplification (MA), resulting in the disregulation of several genes involved in cancer progression, chemotherapy resistance and poor prognosis causing the disregulation of several genes involved in cancer progression and chemotherapy resistance and resulting in a poor prognosis. Moreover, in this contest, therapy-related p53 mutations are frequently found in relapsed cases conferring an even stronger aggressiveness. For this reason, the actual therapy requires new antitumor molecules. Therefore, rapid, accurate, and reproducible preclinical models are needed to evaluate the evolution of the different subtypes and the efficacy of new pharmacological strategies. Procedures. We report the real-time tumorigenesis of MA Neuroblastoma mouse models: transgenic TH-MYCN mice and orthotopic xenograft models with either p53wt or p53mut, by non-invasive micro PET and bioluminescent imaging, respectively. Characterization of MYCN amplification and expression was performed on every collected sample. We tested the efficacy of a new MYCN inhibitor in vitro and in vivo. Results. MicroPET in TH-MYCN mice permitted the identification of Neuroblastoma at an early stage and offered a sensitive method to follow metabolic progression of tumors. The MA orthotopic model harboring multitherapy-related p53 mutations showed a shorter latency and progression and a stronger aggressiveness respect to the p53wt model. The presence of MA and overexpression was confirmed in each model and we saw a better survival in the TH-MYCN homozigous mice treated with the inhibitor. Conclusions. The mouse models obtained show characteristics of non-invasiveness, rapidity and sensitivity that make them suitable for the in vivo preclinical study of MA-NB. In particular, our firstly reported p53mut BLI xenograft orthotopic mouse model offers the possibility to evaluate the role of multitherapy-related p53 mutations and to validate new p53 independent therapies for this highly aggressive Neuroblastoma subtype. Moreover, we have shown potential clinical suitability of an antigene strategy through its cellular and molecular activity, ability to specifically inhibit transcription and in vivo efficacy with no evidence of toxicity.

Epigenetic role of N-Myc in Neuroblastoma

Childhood neuroblastoma is the most common solid tumour of infancy and highly refractory to therapy. One of the most powerful prognostic indicators for this disease is the N-Myc gene amplification, which occurs in approximately 25% of all neuroblastomas. N-Myc is a member of transcription factors belonging to a subclass of the larger group of proteins sharing Basic-Region/Helix–Loop–Helix/Leucin-Zipper (BR/HLH/LZ) motif. N-Myc oncoproteins may determine activation or repression of several genes thanks to different protein-protein interactions that may modulate its transcriptional regulatory ability and therefore its potential for oncogenicity. Chromatin modifications, including histone methylation, have a crucial role in transcription de-regulation of many cancer-related genes. Here, it was investigated whether N-Myc can functionally and/or physically interact with two different factors involved in methyl histone modification: WDR5 (core member of the MLL/Set1 methyltransferase complex) and the de- methylase LSD1. Co-IP assays have demonstrated the presence of both N-Myc-WDR5 and N-Myc-LSD1 complexes in two neuroblastoma cell lines. Human N-Myc amplified cell lines were used as a model system to investigate on transcription activation and/or repression mechanisms carried out by N-Myc-LSD1 and N-Myc-WDR5 protein complexes. qRT-PCR and immunoblot assays underlined the ability of both complexes to positively (N-Myc-WDR5) and negatively (N-Myc-LSD1) influence transcriptional regulation of crititical neuroblastoma N-Myc-related genes, MDM2, p21 and Clusterin. Ch-IP experiments have revealed the binding of the N-Myc complexes above mentioned to the gene promoters analysed. Finally, pharmacological treatment pointed to abolish N-Myc and LSD1 activity were performed to test cellular alterations, such as cell viability and cell cycle progression. Overall, the results presented in this work suggest that N-Myc can interact with two distinct histone methyl modifiers to positively and negatively affect gene transcription in neuroblastoma.

Phase II study of oral capsular 4-hydroxyphenylretinamide (4-HPR/fenretinide) in pediatric patients with refractory or recurrent neuroblastoma: a report from the Children's Oncology Group

To determine the response rate to oral capsular fenretinide in children with recurrent or biopsy proven refractory high-risk neuroblastoma.

RNA interference screening identifies a novel role for autocrine fibroblast growth factor signaling in neuroblastoma chemoresistance

Chemotherapeutic drug resistance is one of the major causes for treatment failure in high-risk neuroblastoma (NB), the most common extra cranial solid tumor in children. Poor prognosis is typically associated with MYCN amplification. Here, we utilized a loss-of-function kinome-wide RNA interference screen to identify genes that cause cisplatin sensitization. We identified fibroblast growth factor receptor 2 (FGFR2) as an important determinant of cisplatin resistance. Pharmacological inhibition of FGFR2 confirmed the importance of this kinase in NB chemoresistance. Silencing of FGFR2 sensitized NB cells to cisplatin-induced apoptosis, which was regulated by the downregulation of the anti-apoptotic proteins BCL2 and BCLX(L). Mechanistically, FGFR2 was shown to activate protein kinase C-δ to induce BCL2 expression. FGFR2, as well as the ligand fibroblast growth factor-2, were consistently expressed in primary NB and NB cell lines, indicating the presence of an autocrine loop. Expression analysis revealed that FGFR2 correlates with MYCN amplification and with advanced stage disease, demonstrating the clinical relevance of FGFR2 in NB. These findings suggest a novel role for FGFR2 in chemoresistance and provide a rational to combine pharmacological inhibitors against FGFR2 with chemotherapeutic agents for the treatment of NB.Oncogene advance online publication, 1 October 2012; doi:10.1038/onc.2012.416.

Novel agents targeting the IGF-1R/PI3K pathway impair cell proliferation and survival in subsets of medulloblastoma and neuroblastoma

The receptor tyrosine kinase (RTK)/phosphoinositide 3-kinase (PI3K) pathway is fundamental for cancer cell proliferation and is known to be frequently altered and activated in neoplasia, including embryonal tumors. Based on the high frequency of alterations, targeting components of the PI3K signaling pathway is considered to be a promising therapeutic approach for cancer treatment. Here, we have investigated the potential of targeting the axis of the insulin-like growth factor-1 receptor (IGF-1R) and PI3K signaling in two common cancers of childhood: neuroblastoma, the most common extracranial tumor in children and medulloblastoma, the most frequent malignant childhood brain tumor. By treating neuroblastoma and medulloblastoma cells with R1507, a specific humanized monoclonal antibody against the IGF-1R, we could observe cell line-specific responses and in some cases a strong decrease in cell proliferation. In contrast, targeting the PI3K p110α with the specific inhibitor PIK75 resulted in broad anti-proliferative effects in a panel of neuro- and medulloblastoma cell lines. Additionally, sensitization to commonly used chemotherapeutic agents occurred in neuroblastoma cells upon treatment with R1507 or PIK75. Furthermore, by studying the expression and phosphorylation state of IGF-1R/PI3K downstream signaling targets we found down-regulated signaling pathway activation. In addition, apoptosis occurred in embryonal tumor cells after treatment with PIK75 or R1507. Together, our studies demonstrate the potential of targeting the IGF-1R/PI3K signaling axis in embryonal tumors. Hopefully, this knowledge will contribute to the development of urgently required new targeted therapies for embryonal tumors.

Neonatal macrocephaly: cerebral primitive neuroectodermal tumor or neuroblastoma as an infrequent cause--a case report and review of the literature

We report a male term newborn presenting with a congenital macrocephaly 3.5 standard deviations above the median, with a wide and tense anterior fontanel, splayed calvarial sutures, and muscular hypotonia. Antenatal head circumferences were repeatedly below the median. A postnatal head ultrasound showed a large right intracerebral mass with right lateral ventricle compression, right temporal horn dilation, and right frontal horn enlargement with lateral displacement. Additional imaging by computed tomography scan and magnetic resonance imaging was performed. A decompression was performed and histology, immunohistochemistry, and molecular biology supported the diagnosis of a primitive neuroectodermal tumor. A MYCN gene amplification assay remained negative. The incidence of neonatal brain tumors is between 1.4 and 4.1/100,000 live births. Their most common presentation is macrocephaly, hydrocephalus, stillbirth, or diagnosis by pre- or postnatal imaging. Although hydrocephaly and intra- or extracranial hemorrhage are the most frequent causes of congenital macrocephaly, this should be initially investigated by head ultrasound. A suspected malignancy will be confirmed by histopathology, immunohistochemistry, and molecular biology.

Effect of peritoneal fluid from endometriosis patients on neuroblastoma cells in culture

AIM: Endometriosis is often associated with lower abdominal pain, dysmenorrhea, dyspareunia, and chronic pelvic pain. There is no correlation between the extent of endometriosis and the intensity of pain. The mechanism of pain in endometriosis is unknown. The aim of our study was to investigate the influence of peritoneal fluid (PF) from endometriosis patients on cultured neural cells that are the morphological basis of nociception, and to determine whether there was a relationship between the rAFS staging and an elevation of TGF-beta1 production by these cells. METHODS: Different human neuroblastoma cell lines were grown to 3/4 confluence and then cultured in presence of PF pooled according to the presence of no, mild, or severe endometriosis. After 6 and 24 h of incubation, the morphological changes were assessed and the metabolic activity was determined. RESULTS: The different cell lines showed strongly varying proliferation and aggregation patterns. The metabolic activity was also varying between cell lines, but no consistently increased cell turnover in the PF when compared with the control medium nor associated to a particular, endometriosis-derived PF pool could be shown. In this experimental setting, we have observed that the cell proliferation in the presence of PF was inhibited, and not enhanced as it might have been expected. Measurement of TGF-beta1 showed higher production rates for this cytokine under exposure to PF than in controls for some but not all tested cell lines, but there was no association with the stage (rAFS) of the disease. CONCLUSION: The neuronal cell culture model may become a useful tool to investigate the endometriosis-derived pain, but different endpoints and cell lines may have to be introduced.

Delineating the mechanism(s) of BDNF/TrkB mediated proliferation in Neuroblastoma

Delineating the mechanism(s) of BDNF/TrkB mediated proliferation in Neuroblastoma Timothy Christopher Graham, B.S. Supervisory Professor: Patrick Zweidler-McKay, MD/PhD Neuroblastoma is the most common extra-cranial solid tumor in children, arising from neural crest precursor cells. The neurotrophin receptors (TrkA/B/C) have been implicated as important prognostic markers, linking the biology of the tumor to patient outcome. High expression of TrkA and TrkC receptors have been linked to favorable biological features and high patient survival, while TrkB is expressed in unfavorable, aggressive tumors. Several studies suggest that high levels and activation of TrkB by its ligand brain-derived neurotrophic factor (BDNF) stimulates tumor cell survival, proliferation, and chemoresistance. However, little is known about the molecular mechanisms that regulate proliferation. The TrkB signaling pathway in neuroblastoma cells has been difficult to evaluate due to the loss of TrkB expression when the cells are used in vitro. Here we determined the role of proximal signaling pathways downstream of TrkB on neuroblastoma proliferation. By analyzing a panel of neuroblastoma cell lines, we found that the SMS-KCN cells express detectable levels of protein and mRNA levels of TrkB as analyzed by western, RT-PCR, and surface expression by flow cytometry. By the addition of exogenous human recombinant BDNF, we showed that activation of TrkB is important in the proliferation of the cells and can be repressed by inhibiting TrkB kinase function. By BDNF stimulation and use of specific kinase inhibitors, the common pathways involving PLCg, PI3K/AKT, and MAPK were initially investigated in addition to PI3K/MTOR and FYN pathways. We demonstrate for the first time that Fyn plays a critical role in TrkB mediated proliferation in neuroblastoma. Constitutively active and over-expressed Fyn reduced neuroblastoma proliferation, as measured by PCNA expression. Knockdown of Fyn by shRNA was shown to cooperate with activated TrkB for an enhanced proliferative response. Although TrkB activation has been implicated in the proliferation of neuroblastoma cells, little is known about its effects on cell cycle regulation. Protein levels of pRB, CDK2, CDK4, CDC25A, cyclin D1, and cyclin E were analyzed following BDNF stimulation. We found that BDNF mediated activation of TrkB induces multiple common proximal signaling pathways including the anti-proliferative Fyn pathway and drives cell cycle machinery to enhance the proliferation of neuroblastoma cells.