cancers Article Neuronal Differentiation-Related Epigenetic Regulator ZRF1 Has Independent Prognostic Value in Neuroblastoma but Is Functionally Dispensable In Vitro Carlos Jiménez 1 , Roberta Antonelli 1 , Marc Masanas 1 , Aroa Soriano 1 , Laura Devis-Jauregui 2,3 , Jessica Camacho 4 , Ainara Magdaleno 1 , Gabriela Guillén 1,5 , Raquel Hladun 1,6 , Luz Jubierre 1 , Josep Roma 1 , David Llobet-Navas 2,3 , Josep Sánchez de Toledo 1,7 , Lucas Moreno 1,6 , Soledad Gallego 1,6 and Miguel F. Segura 1, * 1 Group of Translational Research in Child and Adolescent Cancer, Vall d’Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona (UAB), 08035 Barcelona, Spain; carlos.jimenez@vhir.org (C.J.); roberta.antonelli@vhir.org (R.A.); marc.masanas@vhir.org (M.M.); aroa.soriano@vhir.org (A.S.); ainara.magdaleno@vhir.org (A.M.); gguillen@vhebron.net (G.G.); rhladun@vhebron.net (R.H.); jubierrl@mskcc.org (L.J.); josep.roma@vhir.org (J.R.); jsanchezdetoledo@iconcologia.net (J.S.d.T.); lucas.moreno@vhebron.net (L.M.); sgallego@vhebron.net (S.G.) 2 Molecular Mechanisms and Experimental Therapy in Oncology-Oncobell Program, Bellvitge Biomedical Research Institute (IDIBELL), 08908 L’Hospitalet de Llobregat, Spain; ldevis@idibell.cat (L.D.-J.);  dllobet@idibell.cat (D.L.-N.)  3 Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Instituto de Salud Carlos III, Citation: Jiménez, C.; Antonelli, R.; 28029 Madrid, Spain 4 Pathology Department, Vall d’Hebron University Hospital—UAB, 08035 Barcelona, Spain; Masanas, M.; Soriano, A.; Devis-Jauregui, L.; Camacho, J.; jcamacho@vhebron.net 5 Surgery Department, Vall d’Hebron University Hospital—UAB, 08035 Barcelona, Spain Magdaleno, A.; Guillén, G.; Hladun, 6 Pediatric Oncology and Hematology Department, Vall d’Hebron University Hospital—UAB, R.; Jubierre, L.; et al. Neuronal 08035 Barcelona, Spain Differentiation-Related Epigenetic 7 Catalan Institute of Oncology (ICO), 08908 L’Hospitalet de Llobregat, Spain Regulator ZRF1 Has Independent * Correspondence: miguel.segura@vhir.org Prognostic Value in Neuroblastoma but Is Functionally Dispensable In Simple Summary: Neuroblastoma is the most common pediatric solid tumor occurring outside Vitro. Cancers 2021, 13, 4845. https:// the brain, and it is thought to arise from cells that acquire errors during the normal process of doi.org/10.3390/cancers13194845 embryonal development. Today, we know that embryonal development is regulated by epigenetics, Academic Editors: Luis Franco and a mechanism that determines which genes need to be expressed in each cell type and developmental Rakesh K. Singh step. Epigenetic errors, therefore, are considered contributory to the appearance and progression of tumors such as neuroblastoma. Here, we aimed at finding whether ZRF1, a known epigenetic Received: 26 August 2021 regulator, could play a significant role in the aggressiveness of neuroblastoma. Our results suggest Accepted: 25 September 2021 that ZRF1 does not seem to have any relevant function in neuroblastoma cells; however, the levels of Published: 28 September 2021 this epigenetic regulator are related to the prognostic of neuroblastoma patients and could be used to predict their progression and improve the diagnosis. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Abstract: Neuroblastoma is a pediatric tumor of the peripheral nervous system that accounts for published maps and institutional affil- up to ~15% of all cancer-related deaths in children. Recently, it has become evident that epigenetic iations. deregulation is a relevant event in pediatric tumors such as high-risk neuroblastomas, and a determi- nant for processes, such as cell differentiation blockade and sustained proliferation, which promote tumor progression and resistance to current therapies. Thus, a better understanding of epigenetic factors implicated in the aggressive behavior of neuroblastoma cells is crucial for the development of Copyright: © 2021 by the authors. better treatments. In this study, we characterized the role of ZRF1, an epigenetic activator recruited Licensee MDPI, Basel, Switzerland. to genes involved in the maintenance of the identity of neural progenitors. We combined analysis This article is an open access article of patient sample expression datasets with loss- and gain-of-function studies on neuroblastoma cell distributed under the terms and lines. Functional analyses revealed that ZRF1 is functionally dispensable for those cellular functions conditions of the Creative Commons related to cell differentiation, proliferation, migration, and invasion, and does not affect the cellular Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ response to chemotherapeutic agents. However, we found that high levels of ZRF1 mRNA expression 4.0/). are associated to shorter overall survival of neuroblastoma patients, even when those patients with Cancers 2021, 13, 4845. https://doi.org/10.3390/cancers13194845 https://www.mdpi.com/journal/cancers Cancers 2021, 13, 4845 2 of 16 the most common molecular alterations used as prognostic factors are removed from the analyses, thereby suggesting that ZRF1 expression could be used as an independent prognostic factor in neuroblastoma. Keywords: ZRF1; neuronal differentiation; epigenetics; pediatric cancer; neuroblastoma 1. Introduction Neuroblastoma, a pediatric cancer of the peripheral nervous system, is one of the most common embryonal tumors outside the brain [1]. It is thought to arise from tissues of the sympathoadrenergic system at early stages of embryonic development, specifically from neural crest progenitors that fail to differentiate during dorsolateral migration [2]. From a histopathological perspective, neuroblastoma can be classified based on the grade of morphological differentiation into distinct categories, namely neuroblastoma, ganglioneu- roblastoma, and ganglioneuroma, from less to more differentiated tumors. The last two categories are considered to be benign forms of neuroblastoma [3]. The pathological category can be further classified according to the degree of cellular differentiation. For ex- ample, neuroblastoma can be further classified into undifferentiated, poorly differentiated, and differentiating tumors (reviewed in [4]). The degree of differentiation is part of routine prognostic risk assessment. In fact, high-risk neuroblastomas, which represent up to ~60% of all diagnosed neuroblastomas, are usually undifferentiated or poorly differentiated tumors that present very aggressive behavior and have a five-year overall survival rate below 40% [5]. Pro-differentiation therapies (i.e., with retinoic acid derivatives) are part of the standard of care in neuroblastoma, although they are restricted to the treatment of neuroblastoma minimal residual disease [6]. However, not all patients respond to this treatment. Therefore, a better understanding of the molecular mechanisms that main- tain these types of tumors in an undifferentiated state may reveal new opportunities for therapeutic intervention. Epigenetic regulation (e.g., DNA methylation, histone post-translational modifications, non-coding RNA expression, etc.) is one of the mechanisms that controls the differentiation of neuroblasts (i.e., neuroblastoma precursors). The disruption of the homeostatic epige- netic balance contributes to the developmental arrest of sympathetic progenitors, thereby contributing to neuroblastoma oncogenesis [7]. One of the genes that is determinant in the maintenance of neuronal progenitor identity is zuotin-related factor 1 (ZRF1; also known as MPP11), which is encoded by the DNAJC2 gene [8,9]. ZRF1 belongs to the M-phase phos- phoprotein (MPP) family and was first discovered as a chaperone in the cytosol [10–12]. However, later evidence has shown that it also acts as a chromatin regulator in the nucleus, where it is recruited to ubiquitinated histone H2A at ‘Lys-119’ (H2AK119ub), displacing the polycomb repressor complex 1 (PRC1) from chromatin and facilitating the transcription of neural progenitor-associated genes [13]. In addition to its physiological role in the maintenance of the pluripotency of neural progenitor cells, ZRF1 has already been functionally implicated in cancer. ZRF1 was shown to be oncogenic in solid tumors such as breast [14] and gastric [15] cancers. Furthermore, ZRF1 was shown to be overexpressed in acute myeloid leukemia (AML) acting as a negative regulator of differentiation. In the same study, Demajo and collaborators showed that ZRF1 depletion cooperated with differentiating agents (i.e., retinoic acid) to suppress leukemia in vivo [16]. Thus, owing to the role of ZRF1 of maintaining the undifferentiated state of neural progenitor cells and its functional relevance in the differentiation of some tumors, we sought to determine whether ZRF1 plays a major role in neuroblastoma. Here, we found that the expression of ZRF1 mRNA is increased in advanced disease stages and in tumors, with the most common genetic alterations associated with poor prognosis in neuroblastoma, such as MYCN amplification, gain of chromosome 17q, and loss of 1p36. Moreover, the Cancers 2021, 13, 4845 3 of 16 ZRF1 mRNA level was clearly associated with poor neuroblastoma survival in the absence of other poor-prognosis molecular alterations. However, our gain- and loss-of-function experiments suggest that ZRF1 is neither sufficient nor necessary to sustain the oncogenic properties of neuroblastoma cells, such as cell differentiation, proliferation, or migration. Our results validate ZRF1 as a potential prognostic marker, but discard it as a target for differentiation therapy. 2. Materials and Methods 2.1. Analysis of Neuroblastoma Gene Expression Datasets ZRF1 mRNA expression levels were analyzed from neuroblastoma patient data from the GSE62564, GSE45547 and GSE3960 publicly available datasets. The GSE62564 dataset was used to construct receiver operating characteristic (ROC) curves to determine the prognostic value of ZRF1 expression. The optimal cutoff value was defined according to the Youden index. Overall survival (OS) and the cumulative survival rate were estimated using the Kaplan–Meier method, and the log-rank test was performed to assess differences between groups. Univariate and multivariate Cox proportional hazard regression analyses were used to assess the prognostic significance of ZRF1 on OS. These statistical analyses were performed using the IBM SPSS 21 software. For GSE45547 and GSE3960 datasets, gene expression data was extracted and Kaplan–Meier survival plots were generated using the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl; accessed date: 21 January 2021). ZRF1 mRNA expression levels between different patient groups were analyzed using GraphPad Prism Software (La Jolla, CA, USA), and statistical significance was assessed by Kruskal–Wallis test as a non-parametric ANOVA, and Dunn’s test for multiple comparisons. 2.2. Cell Lines Neuroblastoma cell lines (SK-N-AS, SH-SY5Y, and IMR-32) and embryonic kidney cells (HEK293T) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), the CHLA-90 cell line was purchased from the Children’s Oncology Group Cell Culture and Xenograft Repository (Lubbock, TX, USA), and SK-N-BE(2), and LA1-5s were procured from the Public Health England Culture Collection (Salisbury, UK). Neuroblas- toma cells were cultured and maintained in Iscove’s modified Dulbecco’s medium (Life Technologies, Waltham, MA, USA), supplemented with 20% heat-inactivated fetal bovine serum (South America Premium, Biowest, Nuaillé, France) and 1% insulin-transferrin- selenium supplement (Life Technologies). HEK293T cells were grown in Dulbecco’s modi- fied Eagle’s medium (Life Technologies), supplemented with 10% heat-inactivated fetal bovine serum. Media were supplemented with 100 U/mL penicillin, 100 µg/mL strep- tomycin (Life Technologies), and 5 µg/mL plasmocin (InvivoGen, San Diego, CA, USA). Cultures were maintained at 37 ◦ C in a 5% CO2 saturated atmosphere, and periodically tested for mycoplasma contamination. 2.3. Western Blot Analysis Cells were lysed in RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland). Cell lysates were quantified using a DC protein assay (Bio-Rad, Hercules, CA, USA) and 30 µg of protein was resolved on a 4–12% Tris-glycine sodium dodecyl sulfate polyacrylamide electrophoresis gel (Invitrogen, Carlsbad, CA, USA), then transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked for 1 h with 5% non-fat milk or 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween and probed with primary antibodies overnight at 4 ◦ C. Membranes were incubated with secondary antibodies for 1 h before developing with a chemiluminescent horseradish peroxidase substrate EZ-ECL Chemoluminiscence Detection Kit (Biological Industries, Kibbutz Beit- Haemek, Israel). Protein levels were quantified by densitometry using ImageJ software Cancers 2021, 13, 4845 4 of 16 (National Institutes of Health, Bethesda, MD, USA). Antibodies used for Western blot are listed in Table S1. Original western blot images can be found in Figure S7. 2.4. Proliferation and Colony Formation Assays For the proliferation experiments, transduced or transfected cells were plated at a density of 2–8 × 104 cells /well in 6-well plates and allowed to grow for 7 days with a medium change on day 4. Cells were fixed with 1% glutaraldehyde and stained with 0.5% crystal violet. Stained cells were treated in 15% acetic acid, and the absorbance was read at 590 nm. For the colony formation experiments, cells were plated at a very low density (5–10 × 102 cells/well in 6-well plates) and the medium was changed every 3–4 days. The plates were fixed in glutaraldehyde and stained with crystal violet at day 10 or when colonies were visible to the naked eye. Colonies were photographed and counted using ImageJ software. 2.5. Migration and Invasion Assays For the wound-healing assays, neuroblastoma cells were plated at a density of 3 × 106 cells/well in a 6-well plate. The next day, an artificial wound was created in the confluent cell monolayer. Six predefined fields per condition were photographed under contrast phase microscopy at the indicated time points, and the wound area was measured using Image J software. The migration rate was calculated by normalizing the wound area to time 0. For the transwell invasion assays, 2 × 105 cells were seeded in serum-free media in the upper chamber of 8.0 µm pore size transwells (Corning Life Sciences, Corning, NY, USA) previously coated with a barrier of rat tail collagen I (Corning). The lower chamber was filled with media supplemented with fetal bovine serum. After 16 h, remaining cells were removed from the upper chamber and the cells that migrated to the lower surface of the membrane were fixed with 4% paraformaldehyde, and stained with crystal violet. Invading cells were imaged by bright field microscopy, quantified by diluting crystals in acetic acid, and read at 590 nm. 2.6. Differentiation Assays Neuroblastoma cells were plated at low density (1–1.2 × 105 cells) in collagen-coated 60 mm plates. One day later, cells were treated with 10 µM 13-cis-retinoic acid (Selleckchem, Munich, Germany). Cells were collected at day 5 post-treatment for Western blot analysis. For RARβ gene expression analysis, RNA was extracted from cell lysates using a miRNeasy Mini Kit (Qiagen, Germantown, MA, USA) and retrotranscribed with a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific). Real-time PCR was performed with PerfeCTa SYBR Green Fastmix (Quantabio, Beverly, MA, USA) using L27 as the internal standard. Primers are listed in Table S2. Relative quantification of gene expression was calculated using the 2−DDCt method [17]. For neurite length analyses, 1× 104 cells per well were seeded in collagen-coated glass covers in 24-well plates and treated with retinoic acid for 5 days before fixation with 4% paraformaldehyde. Cells were stained with phalloidin-iFluor 594 (Abcam, Cambridge, UK), following manufacturer’s instructions, and DAPI 10 µ/mL (Invitrogen). Slides were visualized with a FV1000 confocal microscope (Olympus, Shinjuku, Tokyo, Japan). Ten fields were acquired for each biological replicate and processed using ImageJ software. Actin prolongations longer than twice the length of the nucleus (~30 µm) were considered as neurites. 2.7. Vectors and Lentiviral Infection pEV-ZRF1, pCAG-ZRF1, and pLKO with different shZRF1 vectors were kindly pro- vided by Luciano DiCroce. Lentiviruses were generated in HEK293T cells using previously described methods [18,19]. Silent mutations were introduced into the ZRF1 overexpression vector using three sequential site-directed mutagenesis reactions in pCAG-ZRF1, using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA, USA) and checked by Sanger sequencing. Primers used for site-directed mutagenesis and sequencing Cancers 2021, 13, 4845 5 of 16 are listed in Table S2, and the shRNA target sequences are listed in Table S3. After mutage- nesis, the ZRF1 sequence was excised from the pCAG by XhoI digestion and ligated into the FG12 lentiviral overexpression vector. 2.8. ZRF1 Overexpression Experiments Neuroblastoma cells plated at a density of 5 × 105 cells in 60 mm plates were trans- duced with viral supernatant. pEV-transduced cells were selected by separating green fluorescence protein (GFP)-positive cells by fluorescence-assisted cell sorting (FACSAria, BD Biosciences, San Jose, CA, USA) at the Flow Cytometry facility of VHIR. 2.9. ZRF1 Knockdown Experiments For shRNA transduction, 2–8 × 105 cells were plated in 60 mm plates with viral supernatant containing either pLKO-non-silencing control (NSC) or shZRF #1, #2, or #3. After 16 h, the supernatant was replaced with fresh medium, and 24 h later, transduced cells were selected by puromycin resistance (1 µg/mL). Three days after transduction, the cells were detached and used for proliferation experiments. For siRNA knockdown, a set of four pre-designed ON-TARGETplus siRNAs against ZRF1 were purchased from Dharmacon (Lafayette, CO, USA). The siRNA target sequences are listed in Table S3. Neuroblastoma cell lines at a concentration of 1.67 × 105 cells/mL were transfected with siRNA at 25 nM using Lipofectamine 2000 (Life Technologies) following the manufacturer’s instructions. After incubation for 16 h, the medium was replaced. 3. Results 3.1. ZRF1 Is an Independent Prognostic Factor in Neuroblastoma To determine whether ZRF1 is involved in the biology of neuroblastoma, we analyzed publicly available mRNA expression datasets to search for correlations between ZRF1 mRNA levels and different clinicopathological parameters of neuroblastoma patients. A receiver operating characteristic (ROC) curve analysis of a cohort of 498 patients was performed in order to assess a ZRF1 cut-off value (Youden index) that maximized the capacity for overall survival prediction (Table S4). Using this cut-off, patients with higher ZRF1 levels showed a reduction in overall survival when compared to patients with low ZRF1 levels (Figure 1a). ZRF1 was found to be upregulated in MYCN-amplified (MNA) patients, even when patients were split into early (1, 2, 4S) or advanced (3, 4) stages (Figure 1b). ZRF1 levels were also found to be increased in advanced stages of the disease, and this upregulation was maintained even when MNA tumors were excluded (Figure 1c). To verify whether the expression of ZRF1 has prognostic value independent of MYCN amplification or disease stage (both factors intrinsically associated with poor survival), correlation analyses were performed excluding the MNA samples and in the different stages. The results showed that the association between ZRF1 mRNA expression and poor survival remained in non-MNA and low stage tumors (Figure 1d). These findings were validated by contingency analyses (Table S5) and confirmed with two different and independent supplementary neuroblastoma mRNA expression datasets (Figure S1). Finally, univariate and multivariate regression analyses confirmed ZRF1 to be independent prognostic factor of overall survival in neuroblastoma (Figure 1e,f). Next, we analyzed the expression of ZRF1 mRNA in patients with the most common segmental copy alterations associated with neuroblastoma prognosis, including 1p36 loss of heterozygosity (LOH), unbalanced 11q LOH, and unbalanced 17q gain [20,21]. ZRF1 expression levels were found to be higher in patients with 1p36 loss and 17q gain (Figure 2a). A similar trend was also observed in patients with loss of 11q, although the difference was not statistically significant. Of note, the association between ZRF1 expression and poor prognosis was maintained in tumors without these alterations (Figure 2b), further supporting the expression of ZRF1 as an independent prognostic factor. Cancers 2021, 13, 4845 6 of 16 Cancers 2021, 13, x 6 of 16 Figure Figure 1. ZRF1 1. ZRF1 is an is an independent independent prognostic prognostic factor factor in in neuroblastoma.(a) neuroblastoma. (a)Kaplan–Meier Kaplan–Meiersurvival survivalplot plotof ofaacohort cohortof of498 498 patients patients (GSE62564) (GSE62564) split split into into high high and and low low ZRF1 ZRF1 mRNA mRNA expression,based expression, basedononthe theYouden Youdenindex. index.(b) (b)ZRF1 ZRF1mRNA mRNAlevels levels in MYCN-amplified in MYCN-amplified tumors tumors (MNA, (MNA, dark dark red) red) vsvs non-MYCNamplified non-MYCN amplifiedtumors tumors(non-MNA, (non-MNA,light lightred), red),according accordingtotothe the indicated disease stages. (c) ZRF1 mRNA levels according to disease stage in the whole cohort (left) or considering patients indicated disease stages. (c) ZRF1 mRNA levels according to disease stage in the whole cohort (left) or considering patients with non-MNA (middle) or with MNA tumors (right). (d) Kaplan–Meier survival plots comparing samples with high and with non-MNA (middle) or with MNA tumors (right). (d) Kaplan–Meier survival plots comparing samples with high and low ZRF1 levels in the indicated groups of patients. (e) Cox univariate regression analysis of overall survival with different ZRF1 levels in thefeatures. lowclinic-pathological indicated (f)groups of patients.regression Cox multivariate (e) Cox univariate regression analysis of analysisresults overall survival of overall survival confirm ZRF1with different mRNA levels clinic-pathological as an independent features. (f) Cox prognostic multivariate marker regression* analysis in neuroblastoma. means p of overall < 0.05; survival ** means p < results means pZRF1 0.01; ***confirm mRNA < 0.001. levels HR: hazard as an independent prognostic marker in neuroblastoma. * means p < 0.05; *** means p < 0.001. HR: hazard ratio. ratio. Cancers2021, Cancers 13,x4845 2021,13, 7 of1616 7 of ZRF1 Figure2.2.ZRF1 Figure levels levels correlate correlate withwith lowlow survival survival in absence in the the absence of genomic of genomic alterations alterations associated associated withprognosis. with poor poor progno- (a) sis. (a) ZRF1 ZRF1levels mRNA mRNA levels in the in theor presence presence absenceor of absence of thesegmental the indicated indicatedcopy segmental copy(GSE3960, alterations alterations n =(GSE3960, n = 101). 101). (b) Kaplan– Meier survival plotssurvival (b) Kaplan–Meier comparing high plots and low ZRF1 comparing samples high and low from ZRF1the GSE3960 samples fromdataset, in the absence the GSE3960 dataset,orinpresence of theor the absence different segmental copy alterations. ns means non-significant; * means p < 0.05; ** means p < 0.01. presence of the different segmental copy alterations. ns means non-significant; * means p < 0.05; ** means p < 0.01. Analysis AnalysisofofZRF1ZRF1protein proteinexpression expressionby byWestern Westernblot bloton on2424tumor tumorfrozen frozensamples samples from a cohort of 22 neuroblastoma patients was performed to from a cohort of 22 neuroblastoma patients was performed to confirm the mRNA resultsconfirm the mRNA results (FigureS2). (Figure S2).Two Twopairs pairsofofsamples samplescorresponded correspondedtotothe thesame samepatients patientsatatdifferent differentstages stagesofof thedisease: the disease:early early tumor tumor resection resection or orbiopsy, biopsy,and andresection resection of of metastatic metastaticlesions, respectively lesions, respec- (Table tively S6). S6). (Table In most of the In most samples, of the samples, thethe intensity theZRF1 intensityofofthe ZRF1 band band was low lowor orbarely barely detectable,using detectable, usingSK-N-BE(2) SK-N-BE(2)cell cellline linelysates lysatesasaspositive positivecontrol. control.However, However,ititisisinteresting interesting totopoint pointoutoutthat thatthe thecases caseswith withhigher higherexpression ZRF1were expressionofofZRF1 werethose thosecorresponding correspondingtoto relapsed or metastatic neuroblastoma, thus supporting the relapsed or metastatic neuroblastoma, thus supporting the fact that high ZRF1 fact that high ZRF1levels levelsare are present in the most aggressive neuroblastomas. Nevertheless, these present in the most aggressive neuroblastomas. Nevertheless, these results are prelimi- results are preliminary and and nary should be validated should in a in be validated larger cohort a larger of matched cohort neuroblastoma of matched neuroblastoma samples. samples. Sincemost Since mosttumors tumorscontain containheterogeneous heterogeneouscell cellpopulations, populations,including includingmalignant malignantcells, cells, immune cells, fibroblasts, and vascular cells, we proceed to confirm immune cells, fibroblasts, and vascular cells, we proceed to confirm by immunohisto- by immunohistochem- chemistry the ZRF1 istry that that signalsignal the ZRF1 was from was tumor cells. Figure from tumor S3 shows cells. Figure S3 ashows representative image of a representative a neuroblastoma tumor where the stromal component of image of a neuroblastoma tumor where the stromal component of the tumor showsthe tumor shows a weak ZRF1a immunoreactivity while tumor cells are highly positive. weak ZRF1 immunoreactivity while tumor cells are highly positive. InInsummary, summary,ZRF1 ZRF1mRNAmRNAlevels levelsare areassociated associatedwithwithpoorpoorprognosis prognosisininneuroblas- neuroblas- toma and may be used as an independent prognostic marker in the absence ofofMYCN toma and may be used as an independent prognostic marker in the absence MYCN amplificationand amplification andother otherpoor poorprognosis-related prognosis-relatedchromosomal chromosomalaberrations. aberrations. 3.2. ZRF1 Is Not Sufficient to Enhance Neuroblastoma Aggressiveness 3.2. ZRF1 Is not Sufficient to Enhance Neuroblastoma Aggressiveness Given the role of ZRF1 in the regulation of neuronal differentiation and the observed Given the role of ZRF1 in the regulation of neuronal differentiation and the observed correlations in neuroblastoma samples, we studied the functional consequences of ZRF1 correlations in neuroblastoma overexpression in neuroblastomasamples, we studied cell lines. Proteinthe functional expression consequences analysis showedof ZRF1 homoge- neous levels of ZRF1 among a panel of different neuroblastoma cell lines, regardless of Cancers 2021, 13, 4845 8 of 16 MYCN amplification status (Figure 3a). To explore whether increasing the levels of ZRF1 enhanced neuroblastoma aggressiveness, we transduced the SK-N-BE(2) and SK-N-AS neuroblastoma cell lines with a ZRF1 lentiviral overexpression vector (Figure 3b), and then analyzed the effects of ZRF1 on different oncogenic properties. Overexpression of ZRF1 did not enhance proliferation (Figure 3c) or ability to form colonies when cells were plated at a low density (Figure 3d). Drug sensitivity assays were performed against cisplatin, an alkylating agent, and topotecan, an inhibitor of topoisomerase-I, which are two of the neu- Cancers 2021, 13, x roblastoma standard-of-care chemotherapies. However, overexpression9ofof ZRF1 16 did not produce an increased resistance of neuroblastoma cells after 72 h of treatment (Figure 3e). Figure 3. ZRF1Figureoverexpression doesdoes 3. ZRF1 overexpression notnot enhance neuroblastoma enhance neuroblastoma cellaggressiveness. cell line line aggressiveness. (a) expression (a). Left, protein Left, protein anal- expression ysis of and analysis of ZRF1 ZRF1MYCN and MYCN by by Westernblot Western blot of of aa9-neuroblastoma 9-neuroblastoma cell line cellpanel. line Right, panel.densitometry quantification. quantification. Right, densitometry (b) Western blot validation of ZRF1 overexpression in SK-N-BE(2) and SK-N-AS cell lines at 96h post-transduction. (c) Prolif- (b) Western eration blot validation of ZRF1 cell assay of neuroblastoma overexpression in SK-N-BE(2) lines overexpressing ZRF1 compared and SK-N-AS (pEV-empty)-transduced to empty-vector cell lines at 96 h post-transduction. cells. (d) Colony formation assay of neuroblastoma cells overexpressing ZRF1. (c) Proliferation assay of neuroblastoma cell lines overexpressing ZRF1 compared to empty-vectorGraph represents the average of three independ- (pEV-empty)-transduced ent experiments, n = 3 per condition. (e) Cisplatin and topotecan resistance assay of ZRF1-overexpressing cell lines. Cells cells. (d) Colony formation assay of neuroblastoma cells overexpressing ZRF1. Graph represents the average of three were treated for 72 h at the indicated doses, and proliferation was assessed by crystal violet staining. (f) Wound-healing independentassay experiments, n = 3 per in neuroblastoma cells condition. (e)ZRF1. overexpressing Cisplatin Graphsand topotecan represent resistance percentage of ZRF1-overexpressing assayarea of the wound at the indicated times, cell lines. normalized Cells were treated to h for 72 time = 0. (g) at the Invasion doses, indicated assay ofand ZRF1proliferation overexpressingwascellsassessed through a bycollagen barrier crystal for staining. violet 16 h. Invasive (f) cells Wound-healing were detected and quantified by crystal violet staining. (h) Representative microscopic pictures of crystal violet-stained assay in neuroblastoma cells invasive cells. ns means non-significant; ZRF1. overexpressing ** meansGraphs p < 0.01. represent percentage of the wound area at the indicated times, normalized to time = 0. (g) Invasion assay of ZRF1 overexpressing cells through a collagen barrier for 16 h. Invasive cells were detected and quantified by crystal 13-cis-retinoic acid (hereafter violet staining. referred to as (h) Representative RA) is a naturally microscopic picturesoccurring differentiat- of crystal violet-stained ing and therapeutic agent for the treatment of neuroblastoma minimal residual disease invasive cells. ns means non-significant; ** means p < 0.01. Cancers 2021, 13, 4845 9 of 16 Owing to the lineage-conferring migratory capability of neuroblastoma cells, we next explored whether higher levels of ZRF1 alters the migration or the invasion of cells in wound-healing and transwell assays, respectively. ZRF1-transduced cells closed the wound at the same pace as empty vector-infected cells (Figure 3f). Moreover, invasion through a collagen barrier was not affected by ZRF1 overexpression in transwell assays (Figure 3g,h), thereby suggesting that ZRF1 does not modulate the ability of neuroblastoma cells to migrate or invade. 13-cis-retinoic acid (hereafter referred to as RA) is a naturally occurring differentiating and therapeutic agent for the treatment of neuroblastoma minimal residual disease [6,22]. Because ZRF1 has been previously demonstrated to be involved in maintaining neural progenitor stemness [8] and in altering retinoic acid induced differentiation [16], we an- alyzed the effect of modulating ZRF1 in RA-differentiated neuroblastoma cells. After 5 days of RA treatment, ZRF1 levels were found to be decreased 3–4 times in the RA-treated SH-SY5Y and SK-N-BE(2) neuroblastoma cell lines, as compared with vehicle-treated cells (Figure 4a). To evaluate the function of ZRF1 in the process of RA-mediated differentia- tion, neuroblastoma cell lines overexpressing ZRF1 were treated with the differentiating agent and the differentiation outcomes were analyzed. First, ZRF1 overexpression did not rescue or attenuate the decrease in proliferation induced by RA-induced differentiation (Figure 4b); secondly, ZRF1 overexpression did not alter the RA-induced upregulation of the RA receptor RAR-β(Figure 4c); finally, neither the percentage of cells with neurites (Figure 4d,e) nor the neurite length (Figure 4f) were modulated by overexpressing ZRF1. These results indicate that sustained high levels of ZRF1 are not enough to enhance onco- genic properties in neuroblastoma cells or impair RA-mediated neuroblastoma differentiation. 3.3. ZRF1 Knockdown Does Not Impair Neuroblastoma Proliferation and Reveals Inconsistencies between Different Gene Silencing Methodologies As ZRF1 expression was noted in all the neuroblastoma cell lines tested, loss of function experiments represented a good strategy to fully dissect any relevant function of ZRF1 in neuroblastoma cells. Thus, we knocked down ZRF1 using lentiviral vectors. Two different shRNAs targeting the ZRF1 coding region were transduced in six different neuroblastoma cell lines and showed a marked reduction in ZRF1 levels compared to non-silencing control (NSC)-transduced cells (Figures 5a and S4A). In all tested cell lines, shRNA-mediated depletion of ZRF1 significantly reduced the proliferative capacity of neuroblastoma cells (Figures 5b and S4B). Transcriptomic analyses of shRNA-mediated ZRF1 depleted cells showed the involve- ment of several genes related to the cell cycle (Figure S4C,D). When we validated the expression of some of those genes (i.e., AURKB), discrepancies among the molecular effects of the three different shRNAs were observed. For example, while one of the shRNAs (shRNA #1) completely abolished the expression of AURKB, the other two (shRNA #2 and #3) did not (Figure 5c). Cell cycle analyses in ZRF1-depleted cells also showed different profiles. The shRNAs #1 and #2 against ZRF1 showed an increase in the percentage of cells in the G1 phase, whereas shRNA #3 showed a trend towards G2/M arrest (Figure 5d). To discard potential shRNA off-target effects, we performed rescue experiments by overex- pressing a ZRF1 variant with silent mutations in the shRNA target sites (Figure S5). This new mutant ZRF1 was completely insensitive to shRNAs #1 and #3, and partially sensitive to shRNA #2 (Figure 5e). When the phenotypic effects were analyzed, the ectopic expres- sion of ZRF1 did not rescue the reduction in proliferation caused by the transduction of the three different shRNAs (Figure 5f). Thus, we concluded that the phenotypic consequences induced by the different shRNAs were not attributable to ZRF1 depletion. To exclude the possibility that some of the ZRF1 knockdown effects were masked by the shRNA off-targets, we repeated the experiments with small interfering RNA (siRNA). Up to four different siRNA sequences were transfected into neuroblastoma cells. All the sequences reduced the ZRF1 level by more than 85% (Figure 6a). When the phenotypic consequences of siRNA-mediated ZRF1 depletion were analyzed, no differences were found in cell proliferation (Figure 6b), wound healing (Figure 6c), invasion through col- ferentiating agent and the differentiation outcomes were analyzed. First, ZRF1 overex- pression did not rescue or attenuate the decrease in proliferation induced by RA-induced differentiation (Figure 4b); secondly, ZRF1 overexpression did not alter the RA-induced upregulation of the RA receptor RAR-β(Figure 4c); finally, neither the percentage of cells Cancers 2021, 13, 4845 with neurites (Figure 4d,e) nor the neurite length (Figure 4f) were modulated 10 byofoverex- 16 pressing ZRF1. These results indicate that sustained high levels of ZRF1 are not enough to enhance oncogenic properties in neuroblastoma cells or impair RA-mediated neuroblastoma dif- lagen (Figure 6d), or resistance to neuroblastoma therapies such as chemotherapeutics or ferentiation. RA (Figure 6e). . Figure 4. ZRF1 overexpression does not attenuate retinoic acid-induced differentiation. (a) ZRF1 levels after RA-induced Figure 4. ZRF1 overexpression differentiation does notblot. measured by western attenuate retinoic acid-induced Actin-normalized densitometrydifferentiation. quantification of(a) ZRF1 ZRF1 levels levels after beneath is shown RA-induced differentiation measured their respective by western Western blot. blot panels (b)Actin-normalized densitometry Cell proliferation assay quantification of SK-N-BE(2) of ZRF1 levels cells overexpressing ZRF1 is shown treated beneath with their respective RA and normalized versus empty vector-transduced cell treated with vehicle. (c) mRNA levels of the RA-induced RA Western blot panels (b) Cell proliferation assay of SK-N-BE(2) cells overexpressing ZRF1 treated with and normalized versus differentiation empty reporter vector-transduced RAR-β, cell treated assessed by RT-qPCR with vehicle. in SK-N-BE(2) cells. (d)(c) mRNAmicroscopy Confocal levels of the RA-induced representative differenti- images ation of reporter RAR-β, assessed by RT-qPCR in SK-N-BE(2) cells. (d) Confocal microscopy representative ZRF-overexpressing SK-N-BE(2) cells stained with phalloidin and DAPI. (e) Quantification of the number of cells images of ZRF- with neurites (>30 µm prolongations) per field. (f) Average neurite length for each group. ns means non-significant; ** means p < 0.01. different shRNAs targeting the ZRF1 coding region were transduced in six differen roblastoma cell lines and showed a marked reduction in ZRF1 levels compared t silencing control (NSC)-transduced cells (Figure 5a and Figure S4A). In all tested ce Cancers 2021, 13, 4845 shRNA-mediated depletion of ZRF1 significantly reduced the proliferative11 of 16 capa neuroblastoma cells (Figure 5b and Figure S4B). 5. ZRF1 shRNA silencing results in inconsistent and unspecific effects in neuroblastoma cells. (a) ZRF1 expression ure 5. ZRF1Figure shRNA silencing results in inconsistent and unspecific effects in neuroblastoma cells. (a) ZRF1 expressio levels in neuroblastoma cells transduced with two different shRNAs against ZRF1, and a non-silencing control (NSC) as els in neuroblastoma cells transduced with two different shRNAs against ZRF1, and a non-silencing control (NSC) negative control. (b) Cell proliferation in shZRF1-transduced neuroblastoma cell lines compared with those transduced gative control. (b) Cell with NSC, measuredproliferation in shZRF1-transduced by crystal violet. (c) ZRF1 and AURKB levelsneuroblastoma cell lines of shZRF1 transduced compared neuroblastoma with cell lines. (d)those Cell transduc th NSC, measured by crystal cycle analysis violet.72(c) of SK-N-BE(2) ZRF1 and AURKB h post-infection levels by FACS. (e) of shZRF1 Western transduced blot analysis of ZRF1 andneuroblastoma AURKB levels in SK-N- cell lines. (d) C cle analysis of SK-N-BE(2) 72 h post-infection by FACS. (e) Western blot analysis of ZRF1 and AURKB levels in SK-N BE(2) cells overexpressing ZRF1 insensitive to shZRF1, at 96h post-transduction. (f) Cell proliferation assay of SK-N-BE(2) cells overexpressing (2) cells overexpressing ZRF1 insensitive ZRF1 transduced insensitive to shZRF1, with at396h shRNAs against ZRF1. * means post-transduction. (f)p Cell ** means p < 0.01. < 0.05;proliferation assay of SK-N-BE( ls overexpressing insensitive ZRF1 transduced with 3 shRNAs against ZRF1. * means p < 0.05; ** means p < 0.01. Transcriptomic analyses of shRNA-mediated ZRF1 depleted cells showed volvement of several genes related to the cell cycle (Figure S4C,D). When we va the expression of some of those genes (i.e., AURKB), discrepancies among the mo effects of the three different shRNAs were observed. For example, while one shRNAs (shRNA #1) completely abolished the expression of AURKB, the oth (shRNA #2 and #3) did not (Figure 5c). Cell cycle analyses in ZRF1-depleted cel showed different profiles. The shRNAs #1 and #2 against ZRF1 showed an increase percentage of cells in the G1 phase, whereas shRNA #3 showed a trend towards arrest (Figure 5d). To discard potential shRNA off-target effects, we performed res periments by overexpressing a ZRF1 variant with silent mutations in the shRNA sites (Figure S5). This new mutant ZRF1 was completely insensitive to shRNAs #1 a Up to four different siRNA sequences were transfected into neuroblastoma cells. All the sequences reduced the ZRF1 level by more than 85% (Figure 6a). When the phenotypic consequences of siRNA-mediated ZRF1 depletion were analyzed, no differences were found in cell proliferation (Figure 6b), wound healing (Figure 6c), invasion through colla- Cancers 2021, 13, 4845 12 of 16 gen (Figure 6d), or resistance to neuroblastoma therapies such as chemotherapeutics or RA (Figure 6e). Figure Figure 6.6. ZRF1 ZRF1isisdispensable dispensablefor forneuroblastoma neuroblastoma growth, growth, migration, migration, and and drug drug resistance. resistance. (a) (a) ZRF1 ZRF1 levels levels in inneuroblastoma neuroblastoma cells cells transfected with siRNA control and 4 different siZRF1. Actin-normalized densitometry quantification ofofZRF1 transfected with siRNA control and 4 different siZRF1. Actin-normalized densitometry quantification ZRF1 levels levels is is shown beneath their respective ZRF1 knockdown validation Western blot panels (b) Cell viability assay shown beneath their respective ZRF1 knockdown validation Western blot panels (b) Cell viability assay in neuroblastoma in neuroblas- toma cells transfected comparing siControl vs siZRF1 at 96h post-transfection. (c) Wound-healing assay in siZRF1 trans- cells transfected comparing siControl vs siZRF1 at 96h post-transfection. (c) Wound-healing assay in siZRF1 transfected fected neuroblastoma cells at 72 h post-transfection. (d) Invasion assay through a collagen barrier of siZRF1 transfected neuroblastoma cells at 72 h post-transfection. (d) Invasion assay through a collagen barrier of siZRF1 transfected cells cells at 72 h post-transfection. Left, crystal violet quantification of invaded cells. Right, representative images of the inva- at 72assay. sion h post-transfection. Left,topotecan (e) Cisplatin (left), crystal violet quantification (middle), of invaded and retinoic cells. acid (right) Right, representative resistance images of thecell assay of siZRF1-transfected invasion lines. assay.were Cells (e) Cisplatin (left), treated after 72 topotecan (middle), h of transfection and at the retinoicdoses indicated acid (right) for 72 hresistance more andassay of siZRF1-transfected proliferation was assessed bycell lines. crystal Cells were violet treated after 72 h of transfection at the indicated doses for 72 h more and proliferation was assessed by crystal staining. violet staining. In summary, our results indicate that ZRF1 has a prognostic value, but is not function- ally relevant in neuroblastoma cells. 4. Discussion Neuroblastoma is thought to originate from cells of the neural crest that are trans- formed during differentiation and migration toward tissues of the sympathoadrenergic lineage. The occurrence of a transformative event during tissue differentiation dictates the aggressiveness of the tumor. In general, patients with poor prognosis have histologically undifferentiated tumors, whereas those with better prognosis have tumors with histological evidence of cellular differentiation [23]. This differentiation program is tightly regulated by a complex set of signals, including external signaling, activation of specific transcriptional programs, and/or epigenetic events (reviewed in [24]). Experimental results in transgenic mouse models have identified activating ALK mutations and MYCN overexpression as Cancers 2021, 13, 4845 13 of 16 the main oncogenic drivers of neuroblastoma [25,26]. These molecular alterations often converge on mechanisms that block differentiation and confer sustained proliferation ca- pabilities. In particular, genes associated with the maintenance of embryonic and adult stem cells, such as the components of PRC1 or PRC2, have been linked to the initiation and progression of neuroblastoma [27]. For example, BMI1, a core component of the PRC1 complex, has been shown to cooperate in MYCN-driven neuroblastomas by inhibiting cell death and differentiation [28–30]. Focusing on the role of PRC1 in neuronal differentiation, Aloia et al. identified ZRF1 as a transcriptional regulator of neural fates in embryonic stem cells [9]. Furthermore, ZRF1 expression has been associated with poor outcomes in other tumors, such as breast [14,31] or gastric cancer [15], thus suggesting an onco- genic role in cancer. Thus, we sought to determine whether ZRF1 plays a functional role in aggressive neuroblastomas. Data mining of multiple neuroblastoma gene expression datasets confirmed that ZRF1 mRNA expression was elevated in subsets of patients with the most common genetic alterations associated with poor outcomes, thereby supporting our initial hypothesis, although this correlation could not be confirmed at the protein level in neuroblastoma patient samples. Nevertheless, our functional data do not support a relevant contribution of ZRF1 in the oncogenesis of neuroblastoma. Ectopic expression of ZRF1 did not offer proliferation, colony formation, or migratory or invasive advantages to neuroblastoma cells. In acute myeloid leukemia, ZRF1 is a regulator of RA-induced differentiation [16], and because of the relevance of retinoids in neuroblastoma therapy [22], we investigated whether ZRF1 interferes with the response of neuroblastoma cells to RA. When neuroblastoma cells were exposed to 13-cis-RA, a clear reduction in the ZRF1 level was observed, concomitant with an expected reduction in cell proliferation and morphological changes such as neurite outgrowth. However, maintaining high ZRF1 expression ectopically was not sufficient to reverse the RA effect. These results suggest that the downregulation of ZRF1 is a consequence of the RA-induced differentiation process. Although ZRF1 is not sufficient to provide oncogenic advantages, it could still be necessary to maintain the undifferentiated and highly proliferative state of neuroblastoma cells. Previous reports demonstrated that depletion of ZRF1 resulted in a reduction of cell proliferation and the induction of apoptosis in gastric [15] or breast cancer models [14]. Conversely, Kaymak et al. also showed that the reduction in cell proliferation mediated by ZRF1 depletion was accompanied by an increase in the migration and invasion properties of breast cancer cells [31]. Our first set of experiments silencing ZRF1 using two different shRNAs resulted in a marked reduction in cell proliferation in six neuroblastoma cell lines. However, these effects were not rescued by overexpressing the shRNA-insensitive form of ZRF1, thereby indicating that the depletion of ZRF1 was not the causal factor for the reduction in cell proliferation. Consistent with this previous finding, siRNA-mediated depletion of ZRF1 did not alter the proliferative, migratory, or invasive capacities or the sensitivity to RA in neuroblastoma cells, thus confirming that ZRF1 is dispensable for the progression of this type of tumor. It is important to highlight that inconsistencies between different gene silencing methods could have misled the conclusion of this study. However, our use of independent silencing tools and rescue experiments with target protein ectopic expression leaves no room for doubt. Imamura et al. demonstrated that while the effects of ZRF1 on cell proliferation were p53-dependent, those related to migration and invasion were p53-independent [15]. In our work, we covered this aspect by using cell lines with non-functional (CHLA-90, SK-N- BE(2), and SK-N-AS) and functional p53 (SH-SY5Y and IMR-32), and we did not find any differential response in the gain-of-function or loss-of-function experiments. The paradoxical discrepancy between the strong correlation between ZRF1 expression and poor patient outcome, and its dispensable function in tumor cells could be due to one or more of the following reasons: (i) one key characteristic of cancer is uncontrolled transcription. Thus, many genes are likely to be differentially expressed incidentally, rather than reflecting a gene that is driving a biologically significant outcome [32]; (ii) Cancers 2021, 13, 4845 14 of 16 the ZRF1-PRC1 axis may regulate different sets of genes in a lineage-dependent manner; (iii) molecular alterations present in neuroblastoma (i.e., mutation burden, chromosomal copy number variations, etc.) deactivate the physiological regulation of ZRF1-PRC1 on cell proliferation/differentiation programs and become ZRF1 independent; and (iv) the ambivalent molecular function of ZRF1 in neuroblastoma cells may be inclined to a non- essential chaperone role. This hypothesis is supported by our subcellular fractionation analysis in different neuroblastoma cell lines, where ZRF1 was found to be predominantly enriched in the cytosolic fraction (Figure S6). In summary, our data suggest the potential use of ZRF1 expression as an independent prognostic factor, particularly in cases without any other associated molecular prognostic factors. However, ZRF1 does not seem to be a promising target candidate for differentiation therapy for neuroblastoma. 5. Conclusions ZRF1 was found to be an independent prognostic factor of survival in neuroblastoma. However, this correlation cannot be explained by the molecular role of ZRF1 by itself, and it could be the reflection of an underlying molecular mechanism promoting neuroblastoma aggressiveness. Nevertheless, our functional studies highlight the need of proper verifi- cation of shRNA-mediated knockdown experiments through consistent validation with different gene silencing technologies and rescue experiments. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/cancers13194845/s1, Figure S1: ZRF1 is associated to poor prognosis in neuroblastoma. Figure S2: ZRF1 protein levels Western blot analysis in patient tumor samples. Figure S3: ZRF1 protein is detected by immunohistochemistry (IHC) in the tumor cells of a neuroblastoma formalin- fixed paraffin-embedded human sample. Figure S4: Functional and molecular consequences of shRNA-mediated depletion of ZRF1 in neuroblastoma cells. Figure S5: Design of a triple shRNA insensitive ZRF1 overexpression construct. Figure S6: Subcellular localization analysis of ZRF1 by Western blot in a panel of neuroblastoma cell lines. Figure S7. Original Western Blot image of Figures 3a,b, 4a, 5a,c,e, 6a, S2a and S4. Table S1: Antibodies used for Western blot analyses. Table S2: Oligonucleotides used for silent ZRF1 mutagenesis and RARB RT-qPCR. Table S3: ZRF1 targeting sequences. Table S4: Cut-off value for ZRF1 levels analysis, based on the ability for overall survival prediction (using GSE62564, n = 498). Table S5: ZRF1 mRNA expression correlations with clinical variables in neuroblastoma using Fisher’s test (GSE62564, n = 498). Table S6: Neuroblastoma tumor sample clinical data. Author Contributions: Conceptualization, C.J. and M.F.S.; methodology, C.J., R.A., M.M., L.D.-J., J.C., A.M., G.G., L.J. and M.F.S.; bioinformatic analyses, C.J., A.S., L.D.-J., D.L.-N. and M.F.S.; validation, C.J. and M.F.S.; formal analysis, C.J., A.S., R.A. and M.F.S.; investigation C.J. and M.F.S.; data curation, C.J., A.S., L.D.-J., G.G., R.H. and M.F.S.; writing—original draft preparation, C.J. and M.F.S.; writing— review and editing, J.R., D.L.-N., J.S.d.T., L.M. and S.G.; supervision, M.F.S.; funding acquisition J.S.d.T., S.G. and M.F.S. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by Instituto de Salud Carlos III (Grant no. CP16/00006, PI17/00564 and PI20/00530 to M.F Segura and MS17/00063 to D. Llobet-Navas); Asociación Española Contra el Cáncer (LABAE18009SEGU, LABAE19004LLOB); Generalitat de Catalunya AGAUR/European Social Fund (Grant no. 2017FI_B_00095 to C Jiménez); Asociación NEN; Joan Petit foundation; Asociación Pulseras Candela foundation; the #delhospitalalacatedral initiative led by Xavi Vallés; and the Rotary Clubs of Barcelona Eixample, Barcelona Diagonal, Santa Coloma de Gramanet, München-Blutenburg, Deutschland Gemeindienst, and others from Barcelona and its province. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Tumor samples from Vall d’Hebron Hospital neuroblastoma patients are under the registered collection Ref. C.0002311 from Instituto de Salud Carlos III. All patients gave written informed consent. Data Availability Statement: Publicly available datasets were analyzed in this study. These data can be found at http://r2.amc.nl under the accession numbers GSE45547, GSE3960, GSE62564. 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