Comprehensive SNP array study of frequently used neuroblastoma cell lines; copy neutral loss of heterozygosity is common in the cell lines but uncommon in primary tumors

<p>Abstract</p> <p>Background</p> <p>Copy neutral loss of heterozygosity (CN-LOH) refers to a special case of LOH occurring without any resulting loss in copy number. These alterations is sometimes seen in tumors as a way to inactivate a tumor suppressor gene and have been found to be important in several types of cancer.</p> <p>Results</p> <p>We have used high density single nucleotide polymorphism arrays in order to investigate the frequency and distribution of CN-LOH and other allelic imbalances in neuroblastoma (NB) tumors and cell lines. Our results show that the frequency of these near-CN-LOH events is significantly higher in the cell lines compared to the primary tumors and that the types of CN-LOH differ between the groups. We also show that the low-risk neuroblastomas that are generally considered to have a "triploid karyotype" often present with a complex numerical karyotype (no segmental changes) with 2-5 copies of each chromosome. Furthermore a comparison has been made between the three related cell lines SK-N-SH, SH-EP and SH-SY5Y with respect to overall genetic aberrations, and several aberrations unique to each of the cell lines has been found.</p> <p>Conclusions</p> <p>We have shown that the NB tumors analyzed contain several interesting allelic imbalances that would either go unnoticed or be misinterpreted using other genome-wide techniques. These findings indicate that the genetics underlying NB might be even more complex than previously known and that SNP arrays are important analysis tools. We have also showed that these near-CN-LOH events are more frequently seen in NB cell lines compared to NB tumors and that a set of highly related cell lines have continued to evolve secondary to the subcloning event. Taken together our analysis highlights that cell lines in many cases differ substantially from the primary tumors they are thought to represent, and that caution should be taken when drawing conclusions from cell line-based studies.</p

High-resolution array copy number analyses for detection of deletion, gain, amplification and copy-neutral LOH in primary neuroblastoma tumors; Four cases of homozygous deletions of the CDKN2A gene

BACKGROUND: Neuroblastoma is a very heterogeneous pediatric tumor of the sympathetic nervous system showing clinically significant patterns of genetic alterations. Favorable tumors usually have near-triploid karyotypes with few structural rearrangements. Aggressive stage 4 tumors often have near-diploid or near-tetraploid karyotypes and structural rearrangements. Whole genome approaches for analysis of genome-wide copy number have been used to analyze chromosomal abnormalities in tumor samples. We have used array-based copy number analysis using oligonucleotide single nucleotide polymorphisms (SNP) arrays to analyze the chromosomal structure of a large number of neuroblastoma tumors of different clinical and biological subsets. RESULTS: Ninety-two neuroblastoma tumors were analyzed with 50 K and/or 250 K SNP arrays from Affymetrix, using CNAG3.0 software. Thirty percent of the tumors harbored 1p deletion, 22% deletion of 11q, 26% had MYCN amplification and 45% 17q gain. Most of the tumors with 1p deletion were found among those with MYCN amplification. Loss of 11q was most commonly seen in tumors without MYCN amplification. In the case of MYCN amplification, two types were identified. One type displayed simple continuous amplicons; the other type harbored more complex rearrangements. MYCN was the only common gene in all cases with amplification. Complex amplification on chromosome 12 was detected in two tumors and three different overlapping regions of amplification were identified. Two regions with homozygous deletions, four cases with CDKN2A deletions in 9p and one case with deletion on 3p (the gene RBMS3) were also detected in the tumors. CONCLUSION: SNP arrays provide useful tools for high-resolution characterization of significant chromosomal rearrangements in neuroblastoma tumors. The mapping arrays from Affymetrix provide both copy number and allele-specific information at a resolution of 10–12 kb. Chromosome 9p, especially the gene CDKN2A, is subject to homozygous (four cases) and heterozygous deletions (five cases) in neuroblastoma tumors

Omega-3 fatty acids decrease CRYAB, production of oncogenic prostaglandin E-2 and suppress tumor growth in medulloblastoma

Aims: Medulloblastoma (MB) is one of the most common malignant central nervous system tumors of childhood. Despite intensive treatments that often leads to severe neurological sequelae, the risk for resistant relapses remains significant. In this study we have evaluated the effects of the omega 3-long chain polyunsaturated fatty acids (omega 3-LCPUFA) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on MB cell lines and in a MB xenograft model.Main methods: Effects of omega 3-LCPUFA treatment of MB cells were assessed using the following: WST-1 assay, cell death probes, clonogenic assay, ELISA and western blot. MB cells were implanted into nude mice and the mice were randomized to DHA, or a combination of DHA and EPA treatment, or to control group. Treatment effects in tumor tissues were evaluated with: LC-MS/MS, RNA-sequencing and immunohistochemistry, and tumors, erythrocytes and brain tissues were analyzed with gas chromatography.Key findings: omega 3-LCPUFA decreased prostaglandin E2 (PGE(2)) secretion from MB cells, and impaired MB cell viability and colony forming ability and increased apoptosis in a dose-dependent manner. DHA reduced tumor growth in vivo, and both PGE(2) and prostacyclin were significantly decreased in tumor tissue from treated mice compared to control animals. All omega 3-LCPUFA and dihomo-gamma-linolenic acid increased in tumors from treated mice. RNA-sequencing revealed 10 downregulated genes in common among omega 3-LCPUFA treated tumors. CRYAB was the most significantly altered gene and the downregulation was confirmed by immunohistochemistry.Significance: Our findings suggest that addition of DHA and EPA to the standard MB treatment regimen might be a novel approach to target inflammation in the tumor microenvironment

ERBB3 is a marker of a ganglioneuroblastoma/ganglioneuroma-like expression profile in neuroblastic tumours

Background: Neuroblastoma (NB) tumours are commonly divided into three cytogenetic subgroups. However, by unsupervised principal components analysis of gene expression profiles we recently identified four distinct subgroups, r1-r4. In the current study we characterized these different subgroups in more detail, with a specific focus on the fourth divergent tumour subgroup (r4). Methods: Expression microarray data from four international studies corresponding to 148 neuroblastic tumour cases were subject to division into four expression subgroups using a previously described 6-gene signature. Differentially expressed genes between groups were identified using Significance Analysis of Microarray (SAM). Next, gene expression network modelling was performed to map signalling pathways and cellular processes representing each subgroup. Findings were validated at the protein level by immunohistochemistry and immunoblot analyses. Results: We identified several significantly up-regulated genes in the r4 subgroup of which the tyrosine kinase receptor ERBB3 was most prominent (fold change: 132–240). By gene set enrichment analysis (GSEA) the constructed gene network of ERBB3 (n = 38 network partners) was significantly enriched in the r4 subgroup in all four independent data sets. ERBB3 was also positively correlated to the ErbB family members EGFR and ERBB2 in all data sets, and a concurrent overexpression was seen in the r4 subgroup. Further studies of histopathology categories using a fifth data set of 110 neuroblastic tumours, showed a striking similarity between the expression profile of r4 to ganglioneuroblastoma (GNB) and ganglioneuroma (GN) tumours. In contrast, the NB histopathological subtype was dominated by mitotic regulating genes, characterizing unfavourable NB subgroups in particular. The high ErbB3 expression in GN tumour types was verified at the protein level, and showed mainly expression in the mature ganglion cells. Conclusions: Conclusively, this study demonstrates the importance of performing unsupervised clustering and subtype discovery of data sets prior to analyses to avoid a mixture of tumour subtypes, which may otherwise give distorted results and lead to incorrect conclusions. The current study identifies ERBB3 as a clear-cut marker of a GNB/GN-like expression profile, and we suggest a 7-gene expression signature (including ERBB3) as a complement to histopathology analysis of neuroblastic tumours. Further studies of ErbB3 and other ErbB family members and their role in neuroblastic differentiation and pathogenesis are warranted

Rho-associated kinase is a therapeutic target in neuroblastoma

Source at: http://doi.org/10.1073/pnas.1706011114 Neuroblastoma is a peripheral neural system tumor that originates from the neural crest and is the most common and deadly tumor of infancy. Here we show that neuroblastoma harbors frequent mutations of genes controlling the Rac/Rho signaling cascade important for proper migration and differentiation of neural crest cells during neuritogenesis. RhoA is activated in tumors from neuroblastoma patients, and elevated expression of Rho-associated kinase (ROCK)2 is associated with poor patient survival. Pharmacological or genetic inhibition of ROCK1 and 2, key molecules in Rho signaling, resulted in neuroblastoma cell differentiation and inhibition of neuroblastoma cell growth, migration, and invasion. Molecularly, ROCK inhibition induced glycogen synthase kinase 3β-dependent phosphorylation and degradation of MYCN protein. Small-molecule inhibition of ROCK suppressed MYCN-driven neuroblastoma growth in TH-MYCN homozygous transgenic mice and MYCN gene-amplified neuroblastoma xenograft growth in nude mice. Interference with Rho/Rac signaling might offer therapeutic perspectives for high-risk neuroblastoma

Multifocal Neuroblastoma and Central Hypoventilation in An Infant with Germline ALK F1174I Mutation

Funding Information: This work has been supported by grants from the Swedish Cancer Society (TM 15-794; TM 20-1213; PK 19-566), the Swedish Childhood Cancer Foundation (TM 16-147; TM 17-166; TM 19-139; PK 17-122; SF 15-61, 18-99; DT 12-002, NC12-0026), the Swedish Research Council (TM 521-2014-3031), the Swedish state under the LUA/ALF agreement (TM ALFGBG-447171) and the Swedish Foundation for Strategic Research (TM/PK RB13-0204, www.nnbcr.se). SF was the recipient of a Research Assistant Fellowship (14-64), by the Swedish Childhood Cancer Foundation. Publisher Copyright: © 2022 by the authors.A preterm infant with central hypoventilation was diagnosed with multifocal neuroblastoma. Congenital anomalies of the autonomic nervous system in association with neuroblastoma are commonly associated with germline mutations in PHOX2B. Further, the ALK gene is frequently mutated in both familial and sporadic neuroblastoma. Sanger sequencing of ALK and PHOX2B, SNP microarray of three tumor samples and whole genome sequencing of tumor and blood were performed. Genetic testing revealed a germline ALK F1174I mutation that was present in all tumor samples as well as in normal tissue samples from the patient. Neither of the patient’s parents presented the ALK variant. Array profiling of the three tumor samples showed that two of them had only numerical aberrations, whereas one sample displayed segmental alterations, including a gain at chromosome 2p, resulting in two copies of the ALK-mutated allele. Whole genome sequencing confirmed the presence of the ALK variant and did not detect any aberrations in the coding or promotor region of PHOX2B. This study is to our knowledge the first to report a de novo ALK F1174I germline mutation. This may not only predispose to congenital multifocal neuroblastoma but may also contribute to the respiratory dysfunction seen in this patient.Peer reviewe

11q deletion or ALK activity curbs DLG2 expression to maintain an undifferentiated state in neuroblastoma

High-risk neuroblastomas typically display an undifferentiated or poorly differentiated morphology. It is therefore vital to understand molecular mechanisms that block the differentiation process. We identify an important role for oncogenic ALK-ERK1/2-SP1 signaling in the maintenance of undifferentiated neural crest-derived progenitors through the repression of DLG2, a candidate tumor suppressor gene in neuroblastoma. DLG2 is expressed in the murine "bridge signature'' that represents the transcriptional transition state when neural crest cells or Schwann cell precursors differentiate to chromaffin cells of the adrenal gland. We show that the restoration of DLG2 expression spontaneously drives neuroblastoma cell differentiation, high-lighting the importance of DLG2 in this process. These findings are supported by genetic analyses of high-risk 11q deletion neuroblastomas, which identified genetic lesions in the DLG2 gene. Our data also suggest that further exploration of other bridge genes may help elucidate the mechanisms underlying the differentiation of NC-derived progenitors and their contribution to neuroblastomas

The multiple functions of miR-574-5p in the neuroblastoma tumor microenvironment

Neuroblastoma is the most common extracranial solid tumor in childhood and arises from neural crest cells of the developing sympathetic nervous system. Prostaglandin E2 (PGE2) has been identified as a key pro-inflammatory mediator of the tumor microenvironment (TME) that promotes neuroblastoma progression. We report that the interaction between the microRNA miR-574-5p and CUG-binding protein 1 (CUGBP1) induces the expression of microsomal prostaglandin E2 synthase 1 (mPGES-1) in neuroblastoma cells, which contributes to PGE2 biosynthesis. PGE2 in turn specifically induces the sorting of miR-574-5p into small extracellular vesicles (sEV) in neuroblastoma cell lines. sEV are one of the major players in intercellular communication in the TME. We found that sEV-derived miR-574-5p has a paracrine function in neuroblastoma. It acts as a direct Toll-like receptor 7/8 (TLR7/8) ligand and induces α-smooth muscle actin (α-SMA) expression in fibroblasts, contributing to fibroblast differentiation. This is particularly noteworthy as it has an opposite function to that in the TME of lung carcinoma, another PGE2 dependent tumor type. Here, sEV-derived miR-574-5p has an autokrine function that inhibits PGE2 biosynthesis in lung cancer cells. We report that the tetraspanin composition on the surface of sEV is associated with the function of sEV-derived miR-574-5p. This suggests that the vesicles do not only transport miRs, but also appear to influence their mode of action

Identification of epigenetically regulated genes that predict patient outcome in neuroblastoma

<p>Abstract</p> <p>Background</p> <p>Epigenetic mechanisms such as DNA methylation and histone modifications are important regulators of gene expression and are frequently involved in silencing tumor suppressor genes.</p> <p>Methods</p> <p>In order to identify genes that are epigenetically regulated in neuroblastoma tumors, we treated four neuroblastoma cell lines with the demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-dC) either separately or in conjunction with the histone deacetylase inhibitor trichostatin A (TSA). Expression was analyzed using whole-genome expression arrays to identify genes activated by the treatment. These data were then combined with data from genome-wide DNA methylation arrays to identify candidate genes silenced in neuroblastoma due to DNA methylation.</p> <p>Results</p> <p>We present eight genes (<it>KRT19</it>, <it>PRKCDBP</it>, <it>SCNN1A</it>, <it>POU2F2</it>, <it>TGFBI</it>, <it>COL1A2</it>, <it>DHRS3 </it>and <it>DUSP23</it>) that are methylated in neuroblastoma, most of them not previously reported as such, some of which also distinguish between biological subsets of neuroblastoma tumors. Differential methylation was observed for the genes <it>SCNN1A </it>(p < 0.001), <it>PRKCDBP </it>(p < 0.001) and <it>KRT19 </it>(p < 0.01). Among these, the mRNA expression of <it>KRT19 </it>and <it>PRKCDBP </it>was significantly lower in patients that have died from the disease compared with patients with no evidence of disease (fold change -8.3, p = 0.01 for <it>KRT19 </it>and fold change -2.4, p = 0.04 for <it>PRKCDBP</it>).</p> <p>Conclusions</p> <p>In our study, a low methylation frequency of <it>SCNN1A</it>, <it>PRKCDBP </it>and <it>KRT19 </it>is significantly associated with favorable outcome in neuroblastoma. It is likely that analysis of specific DNA methylation will be one of several methods in future patient therapy stratification protocols for treatment of childhood neuroblastomas.</p

Autocrine Prostaglandin E2 Signaling Promotes Tumor Cell Survival and Proliferation in Childhood Neuroblastoma

Background: Prostaglandin E2 (PGE2) is an important mediator in tumor-promoting inflammation. High expression of cyclooxygenase-2 (COX-2) has been detected in the embryonic childhood tumor neuroblastoma, and treatment with COX inhibitors significantly reduces tumor growth. Here, we have investigated the significance of a high COX-2 expression in neuroblastoma by analysis of PGE2 production, the expression pattern and localization of PGE2 receptors and intracellular signal transduction pathways activated by PGE2. Principal Findings: A high expression of the PGE2 receptors, EP1, EP2, EP3 and EP4 in primary neuroblastomas, independent of biological and clinical characteristics, was detected using immunohistochemistry. In addition, mRNA and protein corresponding to each of the receptors were detected in neuroblastoma cell lines. Immunofluorescent staining revealed localization of the receptors to the cellular membrane, in the cytoplasm, and in the nuclear compartment. Neuroblastoma cells produced PGE2 and stimulation of serum-starved neuroblastoma cells with PGE2 increased the intracellular concentration of calcium and cyclic AMP with subsequent phosphorylation of Akt. Addition of 16,16-dimethyl PGE 2 (dmPGE2) increased cell viability in a time, dose- and cell line-dependent manner. Treatment of neuroblastoma cells with a COX-2 inhibitor resulted in a diminished cell growth and viability that was reversed by the addition of dmPGE2. Similarly, PGE 2 receptor antagonists caused a decrease in neuroblastoma cell viability in a dose-dependent manner