Overexpression of H2AFX gene in neuroblastoma is associated with worse prognosis.

BACKGROUND: Neuroblastoma (NB) is the most common solid tumour in childhood, and rises in the sympathetic nervous system. Here, we addressed the in silico analysis of the association between the expression of H2AFX gene involved in DNA damage response, and the survival of a cohort of 786 NB patients. METHODS: In silico gene expression was retrieved from the publicly available dataset summarised by Cangelosi et al., including 13,696 gene expression profiles of 786 NB tumours at onset of disease. The prognostic value of H2AFX (H2A histone family member X) gene expression for event-free survival (EFS) and overall survival (OS) was evaluated by Kaplan-Meier and Cox regression analysis. The main results were validated on another openly accessible in silico database (NRC-283) containing 13,489 gene expressions in 283 NB patients. The expression of H2AFX protein was then tested by immunofluorescence on 48 primary NB samples of different tumour stages. H2AFX activity as an oncogene has been further validated in vitro by silencing the molecule in two NB cell lines, characterised by MYCN amplified or not, and performing cell growth and migration assays. RESULTS: A strong inverse association between H2AFX expression and patients' survival was observed and confirmed by immunofluorescence results on primary NB tissue sections. Cox regression analysis also disclosed H2AFX as an independent predictor of EFS and OS. The gene-silencing experiments strongly suggested an oncogenic role for H2AFX on NB cells, regardless of MYCN amplification. CONCLUSIONS: H2AFX is a prognostic marker for unfavourable NB and could be considered a target for therapeutic interventions.

Neuroblastoma Patients' Outcome and Chromosomal Instability.

Chromosomal instability (CIN) induces a high rate of losses or gains of whole chromosomes or parts of chromosomes. It is a hallmark of most human cancers and one of the causes of aneuploidy and intra-tumor heterogeneity. The present study aimed to evaluate the potential prognostic role of CIN in NB patients at diagnosis. We performed array comparative genomic hybridization analyses on 451 primary NB patients at the onset of the disease. To assess global chromosomal instability with high precision, we focused on the total number of DNA breakpoints of gains or losses of chromosome arms. For each tumor, an array-CGH-based breakpoint instability index (BPI) was assigned which defined the total number of chromosomal breakpoints per genome. This approach allowed us to quantify CIN related to whole genome disruption in all NB cases analyzed. We found differences in chromosomal breakages among the NB clinical risk groups. High BPI values are negatively associated with survival of NB patients. This association remains significant when correcting for stage, age, and MYCN status in the Cox model. Stratified analysis confirms the prognostic effect of BPI index in low-risk NB patients with non-amplified MYCN and with segmental chromosome aberrations.

E2F3 gene expression is a potential negative prognostic marker for localised and MYCN not-amplified neuroblastoma: Results of in silico analysis of 786 samples.

BACKGROUND: Neuroblastoma (NB) is an enigmatic childhood malignancy characterised by a wide range of clinical behaviour. Many potential oncogenes for NB have recently been identified. Among them, E2 transcription factor 3 (E2F3) expression was associated with a poor survival in 134 stage 4S patients, but evidence for other stage groups remains poorly investigated. METHODS: We have analysed the expression of E2F3 gene from a database of 786 NB samples. Overall and event-free survivals (EFS) were assessed by the Kaplan-Meier method, splitting the data on the median and tertile expression values. The Cox model was applied to control for the confounding by stage, age and MYCN amplification. Validation was performed by an in silico analysis of an independent cohort of 283 NB patients. Furthermore, an immunofluorescence analysis on 48 formalin-fixed, paraffin-embedded NB specimens was also performed. RESULTS: E2F3 overexpression was associated with a poor survival (EFS = 84%, 95% CI: 79%-95%, for low expression levels; EFS = 62%, 95% CI: 56%-68% for middle levels; EFS = 30%, 95% CI: 24%-36%, for high levels, p < .001). This association was confirmed in multivariable analysis and was more evident in patients with MYCN not-amplified and localised stages. Immunofluorescence results and the validation on an independent cohort of NB primary samples confirmed these findings. CONCLUSIONS: E2F3 is a new potential prognostic marker in NB with favourable characteristics at diagnosis. Further studies are needed to elucidate the potential role of E2F3 in NB oncogenesis and progression, in order to identify new targets for therapeutic interventions.

Genomic Analysis Made It Possible to Identify Gene-Driver Alterations Covering the Time Window between Diagnosis of Neuroblastoma 4S and the Progression to Stage 4.

Neuroblastoma (NB) is a tumor of the developing sympathetic nervous system. Despite recent advances in understanding the complexity of NB, the mechanisms that determine its regression or progression are still largely unknown. Stage 4S NB is characterized by a favorable course of disease and often by spontaneous regression, while progression to true stage 4 is a very rare event. Here, we focused on genomic analysis of an NB case that progressed from stage 4S to stage 4 with a very poor outcome. Array-comparative genomic hybridization (a-CGH) on tumor-tissue DNA, and whole-exome sequencing (WES) on exosomes DNA derived from plasma collected at the onset and at the tumor progression, pointed out relevant genetic changes that can explain this clinical worsening. The combination of a-CGH and WES data allowed for the identification iof somatic copy number aberrations and single-nucleotide variants in genes known to be responsible for aggressive NB. KLRB1, MAPK3 and FANCA genes, which were lost at the time of progression, were studied for their possible role in this event by analyzing in silico the impact of their expression on the outcome of 786 NB patients.

Exosomes from Plasma of Neuroblastoma Patients Contain Doublestranded DNA Reflecting the Mutational Status of Parental Tumor Cells.

Neuroblastoma (NB) is an aggressive infancy tumor, leading cause of death among preschool age diseases. Here we focused on characterization of exosomal DNA (exo-DNA) isolated from plasma cell-derived exosomes of neuroblastoma patients, and its potential use for detection of somatic mutations present in the parental tumor cells. Exosomes are small extracellular membrane vesicles secreted by most cells, playing an important role in intercellular communications. Using an enzymatic method, we provided evidence for the presence of double-stranded DNA in the NB exosomes. Moreover, by whole exome sequencing, we demonstrated that NB exo-DNA represents the entire exome and that it carries tumor-specific genetic mutations, including those occurring on known oncogenes and tumor suppressor genes in neuroblastoma (ALK, CHD5, SHANK2, PHOX2B, TERT, FGFR1, and BRAF). NB exo-DNA can be useful to identify variants responsible for acquired resistance, such as mutations of ALK, TP53, and RAS/MAPK genes that appear in relapsed patients. The possibility to isolate and to enrich NB derived exosomes from plasma using surface markers, and the quick and easy extraction of exo-DNA, gives this methodology a translational potential in the clinic. Exo-DNA can be an attractive non-invasive biomarker for NB molecular diagnostic, especially when tissue biopsy cannot be easily available.

Genomic coamplification of CDK4/MDM2/FRS2 is associated with very poor prognosis and atypical clinical features in neuroblastoma patients.

Neuroblastoma (NB) is the most common extracranial malignant tumor of childhood and is characterized by a broad heterogeneity in clinical presentation and evolution. Recent advances in pangenomic analysis of NB have revealed different recurrent chromosomal aberrations. Indeed, it is now well established that the overall genomic profile is important for treatment stratification. In previous studies, 11 genes were shown to be recurrently amplified (ODC1, ALK, GREB1, NTSR2, LIN28B, MDM2, CDK4, MYEOV, CCND1, TERT, and MYC) besides MYCN, with poor survival of NB patients harboring these amplifications being suggested. Genomic profiles of 628 NB samples analyzed by array-comparative genome hybridization (a-CGH) were re-examined to identify gene amplifications other them MYCN amplification. Clinical data were retrospectively collected. We additionally evaluated the association of FRS2 gene expression with NB patient outcome using the public R2 Platform. We found eight NB samples with high grade amplification of one or two loci on chromosome arm 12q. The regional amplifications were located on bands 12q13.3-q14.1 and 12q15-q21.1 involving the genes CDK4, MDM2, and the potential oncogenic gene FRS2. The CDK4, MDM2, and FRS2 loci were coamplified in 8/8 samples. The 12q amplifications were associated with very poor prognosis and atypical clinical features of NB patients. Further functional and clinical investigations are needed to confirm or refute these associations.

Loss of whole chromosome X predicts prognosis of neuroblastoma patients with numerical genomic profile.

BACKGROUND: Neuroblastoma (NB), a pediatric tumor of the sympathetic nervous system, is characterized by very frequent chromosomal aberrations at the onset of the disease. Identification of further risk factors for relapse, which could lead to increased survival and potentially reduced late effects among survivors, is still urgently needed. Segmental chromosome aberrations (SCA) are associated with poor prognosis, whereas numerical whole-chromosome aberrations (NCA) are found in patients with a good prognosis; however, a small percentage of the latter patients (10%-15%) subsequently relapse and/or die of disease. PROCEDURE: DNA copy-number data from 174 NB patients with an NCA genomic profile were analyzed. Association between NCA and event-free survival (EFS) was investigated by the Kaplan-Meier estimator and prognostic decision tree (DT). RESULTS: DT identified 65 patients with normal chromosome X and an excellent five-year EFS (100%) independently from the stage at diagnosis. The association between poor EFS and whole chromosome X alterations was confirmed after stratification into two groups of different expected prognosis and by internal validation via bootstrap analysis. Furthermore, the association was also observed in an independent cohort of NB patients extracted from the data set of the National Cancer Institute TARGET Project for Neuroblastoma, but sample size was small (n = 75) and statistical significance was not achieved. CONCLUSIONS: Loss of whole chromosome X may represent a new prognostic marker for NB patients with an NCA genomic profile. If confirmed by further studies, this finding could indicate that such patients should be reclassified as intermediate risk and treated accordingly.

Role of GOLPH3 and TPX2 in Neuroblastoma DNA Damage Response and Cell Resistance to Chemotherapy.

Neuroblastoma (NB) is an aggressive, relapse-prone infancy tumor of the sympathetic nervous system and is the leading cause of death among preschool age diseases, so the search for novel therapeutic targets is crucial. Golgi phosphoprotein 3 (GOLPH3) has been reported to be involved in the development, and in the DNA damage response, of various human cancers. Golgi dispersal is a common feature of DNA damage response in mammalian cells. Understanding how cells react to DNA damage is essential in order to recognize the systems used to escape from elimination. We induced DNA damage in two human neuroblastoma cell lines by curcumin. The exposure of neuroblastoma cells to curcumin induced: (a) up-regulation of GOLPH3+ cells; (b) augmentation of double-strand breaks; (c) Golgi fragmentation and dispersal throughout the cytoplasm; (d) increase of apoptosis and autophagy; (e) increased expression of TPX2 oncoprotein, able to repair DNA damage. Primary neuroblastoma samples analysis confirmed these observations. Our findings suggest that GOLPH3 expression levels may represent a clinical marker of neuroblastoma patients' responsiveness to DNA damaging therapies-and of possible resistance to them. Novel molecules able to interfere with GOLPH3 and TPX2 pathways may have therapeutic benefits when used in combination with standard DNA damaging therapeutic agents in neuroblastoma.

Telomere shortening and increased oxidative stress are restricted to venous tissue in patients with varicose veins: A merely local disease?

Shortened telomere length (TL) and oxidative stress have been described in several vascular disorders at both the tissue and circulating level. However, to our knowledge, there are no reports about TL associated with varicose vein (VV) disease. This paper aimed to evaluate, at the tissue and circulating level, TL and oxidative stress in VV disease, compared to the corresponding counterparts from abdominal aortic aneurysm (AAA) patients and control healthy subjects. TL was measured using quantitative fluorescence in situ hybridization (Q-FISH). Oxidative stress was evaluated by measuring the malondialdehyde (MDA) concentration by thiobarbituric acid reactive substance/s (TBARS) assay. At the vascular tissue level, VV patients had shortened TL and a high MDA concentration, similarly to AAA patients. Conversely, blood lymphocytes and epidermal cells from VV patients had a TL similar to healthy controls and significantly longer than the same cells from AAA patients. Moreover, the MDA concentration in plasma from VV patients was significantly lower than from the AAA group. Linear regression analysis showed a statistically significant inverse correlation between the blood lymphocyte TL and plasma MDA level. Our results suggest that, unlike AAA, telomere attrition in VV tissue is not a systemic phenomenon but it may be attributable to tissue microenvironment conditions and possibly to high local oxidative stress.

A Simple, Test-Based Method to Control the Overestimation Bias in the Analysis of Potential Prognostic Tumour Markers.

The early evaluation of prognostic tumour markers is commonly performed by comparing the survival of two groups of patients identified on the basis of a cut-off value. The corresponding hazard ratio (HR) is usually estimated, representing a measure of the relative risk between patients with marker values above and below the cut-off. A posteriori methods identifying an optimal cut-off are appropriate when the functional form of the relation between the marker distribution and patient survival is unknown, but they are prone to an overestimation bias. In the presence of a small sample size, which is typical of rare diseases, the external validation sets are hardly available and internal cross-validation could be unfeasible. We describe a new method to obtain an unbiased estimate of the HR at an optimal cut-off, exploiting the simple relation between the HR and the associated p-value estimated by a random permutation analysis. We validate the method on both simulated data and set of gene expression profiles from two large, publicly available data sets. Furthermore, a reanalysis of a previously published study, which included 134 Stage 4S neuroblastoma patients, allowed for the identification of E2F1 as a new gene with potential oncogenic activity. This finding was confirmed by an immunofluorescence analysis on an independent cohort.

Multiple Genes with Potential Tumor Suppressive Activity Are Present on Chromosome 10q Loss in Neuroblastoma and Are Associated with Poor Prognosis.

Neuroblastoma (NB) is a tumor affecting the peripheral sympathetic nervous system that substantially contributes to childhood cancer mortality. Despite recent advances in understanding the complexity of NB, the mechanisms determining its progression are still largely unknown. Some recurrent segmental chromosome aberrations (SCA) have been associated with poor survival. However, the prognostic role of most SCA has not yet been investigated. We examined a cohort of 260 NB primary tumors at disease onset for the loss of chromosome 10q, by array-comparative genomic hybridization (a-CGH) and Single Nucleotide Polymorphism (SNP) array and we found that 26 showed 10q loss, while the others 234 displayed different SCA. We observed a lower event-free survival for NB patients displaying 10q loss compared to patients with tumors carrying other SCA. Furthermore, analyzing the region of 10q loss, we identified a cluster of 75 deleted genes associated with poorer outcome. Low expression of six of these genes, above all CCSER2, was significantly correlated to worse survival using in silico data from 786 NB patients. These potential tumor suppressor genes can be partly responsible for the poor prognosis of NB patients with 10q loss.

Treatment of newborn G6pc(-/-) mice with bone marrow-derived myelomonocytes induces liver repair.

BACKGROUND & AIMS: Several studies have shown that bone marrow-derived committed myelomonocytic cells can repopulate diseased livers by fusing with host hepatocytes and can restore normal liver function. These data suggest that myelomonocyte transplantation could be a promising approach for targeted and well-tolerated cell therapy aimed at liver regeneration. We sought to determine whether bone marrow-derived myelomonocytic cells could be effective for liver reconstitution in newborn mice knock-out for glucose-6-phosphatase-α. METHODS: Bone marrow-derived myelomonocytic cells obtained from adult wild type mice were transplanted in newborn knock-out mice. Tissues of control and treated mice were frozen for histochemical analysis, or paraffin-embedded and stained with hematoxylin and eosin for histological examination or analyzed by immunohistochemistry or fluorescent in situ hybridization. RESULTS: Histological sections of livers of treated knock-out mice revealed areas of regenerating tissue consisting of hepatocytes of normal appearance and partial recovery of normal architecture as early as 1 week after myelomonocytic cells transplant. FISH analysis with X and Y chromosome paints indicated fusion between infused cells and host hepatocytes. Glucose-6-phosphatase activity was detected in treated mice with improved profiles of liver functional parameters. CONCLUSIONS: Our data indicate that bone marrow-derived myelomonocytic cell transplant may represent an effective way to achieve liver reconstitution of highly degenerated livers in newborn animals.

Molecular Genetics in Neuroblastoma Prognosis.

In recent years, much research has been carried out to identify the biological and genetic characteristics of the neuroblastoma (NB) tumor in order to precisely define the prognostic subgroups for improving treatment stratification. This review will describe the major genetic features and the recent scientific advances, focusing on their impact on diagnosis, prognosis, and therapeutic solutions in NB clinical management.

High Grade of Amplification of Six Regions on Chromosome 2p in a Neuroblastoma Patient with Very Poor Outcome: The Putative New Oncogene TSSC1.

We observed a case of high-risk neuroblastoma (NB) carried by a 28-month-old girl, displaying metastatic disease and a rapid decline of clinical conditions. By array-CGH analysis of the tumor tissue and of the metastatic bone marrow aspirate cells, we found a high-grade amplification of six regions besides MYCN on bands 2p25.3-p24.3. The genes involved in these amplifications were MYT1L, TSSC1, CMPK2, RSAD2, RNF144A, GREB1, NTSR2, LPIN1, NBAS, and the two intergenic non-protein coding RNAs LOC730811 and LOC339788. We investigated if these DNA co-amplifications may have an effect on enhancing tumor aggressiveness. We evaluated the association between the high expression of the amplified genes and NB patient's outcome using the integration of gene expression data of 786 NB samples profiled with different public platforms from patients with at least five-year follow-up. NB patients with high expression of the TSSC1 gene were associated with a reduced survival rate. Immunofluorescence staining on primary tumor tissues confirmed that the TSSC1 protein expression was high in the relapsed or dead stage 4 cases, but it was generally low in NB patients in complete remission. TSSC1 appears as a putative new oncogene in NB.

Enhancement of Neuroblastoma NK-Cell-Mediated Lysis through NF-kB p65 Subunit-Induced Expression of FAS and PVR, the Loss of Which Is Associated with Poor Patient Outcome.

High-risk neuroblastoma (NB) is a rare childhood cancer whose aggressiveness is due to a variety of chromosomal genetic aberrations, including those conferring immune evasion. Indeed, NB cells adopt several molecular strategies to evade recognition by the immune system, including the downregulation of ligands for NK-cell-activating receptors. To date, while molecular strategies aimed at enhancing the expression of ligands for NKG2D- and DNAM-1-activating receptors have been explored, no evidence has been reported on the immunomodulatory mechanisms acting on the expression of death receptors such as Fas in NB cells. Here, we demonstrated that transient overexpression of the NF-kB p65 subunit upregulates the surface expression of Fas and PVR, the ligand of DNAM-1, thus making NB cell lines significantly more susceptible to NK-cell-mediated apoptosis, recognition, and killing. In contrast, IFNγ and TNFα treatment, although it induced the upregulation of FAS in NB cells and consequently enhanced NK-cell-mediated apoptosis, triggered immune evasion processes, including the strong upregulation of MHC class I and IDO1, both of which are involved in mechanisms leading to the impairment of a proper NK-cell-mediated killing of NB. In addition, high-resolution array CGH analysis performed in our cohort of NB patients revealed that the loss of FAS and/or PVR genes correlated with low survival independently of the disease stage. Our data identify the status of the FAS and PVR genes as prognostic biomarkers of NB that may predict the efficacy of NK-cell-based immunotherapy of NB. Overall, restoration of surface expression of Fas and PVR, through transient upregulation of NF-kB, may be a clue to a novel NK-cell-based immunotherapy of NB.

Nutlin-3a Enhances Natural Killer Cell-Mediated Killing of Neuroblastoma by Restoring p53-Dependent Expression of Ligands for NKG2D and DNAM-1 Receptors.

In this study, we explored whether Nutlin-3a, a well-known, nontoxic small-molecule compound antagonizing the inhibitory interaction of MDM2 with the tumor suppressor p53, may restore ligands for natural killer (NK) cell-activating receptors (NK-AR) on neuroblastoma cells to enhance the NK cell-mediated killing. Neuroblastoma cell lines were treated with Nutlin-3a, and the expression of ligands for NKG2D and DNAM-1 NK-ARs and the neuroblastoma susceptibility to NK cells were evaluated. Adoptive transfer of human NK cells in a xenograft neuroblastoma-bearing NSG murine model was assessed. Two data sets of neuroblastoma patients were explored to correlate p53 expression with ligand expression. Luciferase assays and chromatin immunoprecipitation analysis of p53 functional binding on PVR promoter were performed. Primary neuroblastoma cells were also treated with Nutlin-3a, and neuroblastoma spheroids obtained from one high-risk patient were assayed for NK-cell cytotoxicity. We provide evidence showing that the Nutlin-3a-dependent rescue of p53 function in neuroblastoma cells resulted in (i) increased surface expression of ligands for NK-ARs, thus rendering neuroblastoma cell lines significantly more susceptible to NK cell-mediated killing; (ii) shrinkage of human neuroblastoma tumor masses that correlated with overall survival upon adoptive transfer of NK cells in neuroblastoma-bearing mice; (iii) and increased expression of ligands in primary neuroblastoma cells and boosting of NK cell-mediated disaggregation of neuroblastoma spheroids. We also found that p53 was a direct transcription factor regulating the expression of PVR ligand recognized by DNAM-1. Our findings demonstrated an immunomodulatory role of Nutlin-3a, which might be prospectively used for a novel NK cell-based immunotherapy for neuroblastoma.

The combined therapeutic effects of bortezomib and fenretinide on neuroblastoma cells involve endoplasmic reticulum stress response.

PURPOSE: The proteasome inhibitor bortezomib inhibited cell growth and angiogenesis in neuroblastoma. Bortezomib has been shown to induce synergistic activity when combined with other antineoplastic agents. Here we have investigated the antitumor activity of bortezomib in combination with fenretinide, a synthetic retinoid, against neuroblastoma cells. EXPERIMENTAL DESIGN: Different neuroblastoma cell lines were tested for sensitivity to bortezomib and fenretinide, given alone or in different dose-dependent and time-dependent combination schedules. Cell proliferation, cell viability, and apoptosis were evaluated by measuring 3H-thymidine incorporation, trypan blue staining, DNA fragmentation, and western blot analysis. Angiogenesis was assessed by the chick embryo chorioallantoic membrane assay. An orthotopic neuroblastoma mouse model was used to examine in vivo sensitivity. RESULTS: Each compound alone was able to induce a dose-dependent inhibition of cell proliferation, with a significant enhanced antiproliferative effect for the drugs used in combination. This inhibition was characterized by marked G2-M and G1 cell cycle arrest with nearly complete depletion of S phase. Bortezomib and fenretinide in association triggered an increased apoptosis through activation of specific genes of the endoplasmic reticulum stress compared with either drug tested alone. Tumor-bearing mice treated with bortezomib plus fenretinide lived statistically significantly longer than mice treated with each drug alone. Histologic evaluation and chorioallantoic membrane analysis of primary tumors showed that the combined therapeutic activity of bortezomib and fenretinide rested upon antitumor and antiangiogenic mechanisms. CONCLUSIONS: These findings provide the rationale for the development of a new therapeutic strategy for neuroblastoma based on this pharmacologic combination.

Roniciclib down-regulates stemness and inhibits cell growth by inducing nucleolar stress in neuroblastoma.

Neuroblastoma, an embryonic tumor arising from neuronal crest progenitor cells, has been shown to contain a population of undifferentiated stem cells responsible for the malignant state and the unfavorable prognosis. Although many previous studies have analyzed neuroblastoma stem cells and their therapeutic targeting, this topic appears still open to novel investigations. Here we found that neurospheres derived from neuroblastoma stem-like cells showed a homogeneous staining for several key nucleolar proteins, such as Nucleolin, Nucleophosmin-1, Glypican-2 and PES-1. We investigated the effects of Roniciclib (BAY 1000394), an anticancer stem cells agent, on neurospheres and on an orthotopic neuroblastoma mouse model, discovering an impressive inhibition of tumor growth and indicating good chances for the use of Roniciclib in vivo. We demonstrated that Roniciclib is not only a Wnt/ß-catenin signaling inhibitor, but also a nucleolar stress inducer, revealing a possible novel mechanism underlying Roniciclib-mediated repression of cell proliferation. Furthermore, we found that high expression of Nucleophosmin-1 correlates with patients' short survival. The co-expression of several stem cell surface antigens such as CD44v6 and CD114, together with the nucleolar markers here described, extends new possibilities to isolate undifferentiated subpopulations from neuroblastoma and identify new targets for the treatment of this childhood malignancy.

Ezrin interacts with the tumor suppressor CHL1 and promotes neuronal differentiation of human neuroblastoma.

In a previous study, we demonstrated that CHL1, the neuronal cell adhesion molecule close homolog of L1, acts as a tumor suppressor in human neuroblastoma (NB), a still highly lethal childhood malignancy, influencing its differentiation and proliferation degree. Here we found that ezrin, one of the ERM (ezrin, radixin, moesin) proteins involved in cytoskeleton organization, strongly interacts with CHL1. The low expression of EZRIN, as well as the low expression of CHL1 and of the neuronal differentiation marker MAP2, correlates with poor outcome in NB patients. Knock-down of ezrin in HTLA-230 cell line induces neurite retraction, enhances cell proliferation and migration, and triggers anchorage-independent growth, with effects very similar to those already obtained by CHL1 silencing. Furthermore, lack of ezrin inhibits the expression of MAP2 and of the oncosuppressor molecule p53, whereas it enhances MAPK activation, all typical features of tumor aggressiveness. As already described, CHL1 overexpression in IMR-32 cell line provokes an opposite trend, but the co-silencing of ezrin reduces these effects, confirming the hypothesis that CHL1 acts in close connection with ezrin. Overall, our data show that ezrin reinforces the differentiating and oncosuppressive functions of CHL1, identifying this ERM protein as a new targetable molecule for NB therapy.

The Over-Expression of E2F3 Might Serve as Prognostic Marker for Neuroblastoma Patients with Stage 4S Disease.

Stage 4S neuroblastoma is a childhood cancer occurring in infants (<12 months at diagnosis) with metastases limited to liver, skin, and bone marrow (<10%). It is associated with an excellent outcome, due to its notable ability to undergo spontaneous regression without any therapeutic intervention. However, a subgroup of patients is doomed to relapse and eventually to die in spite of aggressive therapies. Stage 4S neuroblastoma shows characteristic hypermethylation of genes involved in the telomere maintenance, indicating that the dysregulation of these genes might serve as prognostic marker. The retinoblastoma tumor suppressor protein (RB)-E2F transcription factors pathway is one of the critical tumor-suppressor/oncogene pathways involved in regulating telomerase expression. We have interrogated in silicopublic neuroblastoma databases for regulators involved in the RB-E2F pathway especially for E2F factors themselves, and we identified the E2F transcription factor 3 (E2F3) expression as a potential prognostic marker in stage 4S neuroblastoma. In order to confirm this finding, we screened 38 paraffin-embedded tissue samples stage 4S neuroblastoma for E2F3 protein expression using immunofluorescence, and we observed that augmented expression was strongly associated with impaired event-free survival. These results indicate that E2F3 expression might serve as prognostic marker in patients with stage 4S disease.