Identification of recurrent 3q13.31 chromosomal rearrangement indicates LSAMP as a tumor suppressor gene in neuroblastoma
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- Published online on: January 3, 2023 https://doi.org/10.3892/ijo.2023.5475
- Article Number: 27
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Copyright: © Martinez-Monleon et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Neuroblastoma (NB) is a pediatric neuroendocrine tumor of the sympathetic nervous system. NB arises from undifferentiated neural crest precursor cells, which results in tumors emerging mainly in the adrenal medulla or paraspinal ganglia. It is the most common malignancy diagnosed in the first year of life and the most common extracranial solid tumor in childhood (1). NB represents 8-10% of all pediatric tumors, but results in ~15% of all pediatric cancer deaths (2,3). Tumors commonly arise in children <5 years of age with an average age of 18 months at the time of diagnosis (4). Understanding NB is a challenge due to the heterogeneity in the course of the disease, ranging from tumors that show spontaneous regression (even without treatment) to highly aggressive tumors with a treatment-resistant phenotype. Patients with high-risk NB (HR-NB) have a very poor prognosis, with <50% 5-year overall survival rate despite heavy multimodal treatment, and the survival rate is <10% for relapsed cases (5). Considering the high mortality rate associated with HR-NB, increased understanding of the biology behind this malignancy is important as it may lead to improvement in patient stratification and ultimately in novel therapeutic strategies.
Analysis of chromosomal copy number variations (CNVs) in NB provides relevant information for the diagnosis and prognosis of the disease. Tumors with numerical aberrations only (gain/loss of whole chromosomes) and near triploid karyotype are typically associated with a good prognosis, whereas tumors with segmental rearrangements and near di- or tetraploid karyotypes generally have a poor prognosis (6,7). Recurrent CNVs in NB include loss of chromosome 1p, 3p, 4p and 11q, gain of 1q, 2p and 17q, and amplification of the MYCN oncogene; notably, MYCN amplification and 11q-deletion are associated with more aggressive NB. Small focal alterations have been shown to target cell cycle genes (e.g., CDKN2A/B, CCND1, CDK4/6 andMDM2) (8-10) or genes involved in chromatin remodeling and telomere maintenance (e.g., ATRX, ARID1A/ARID1B andTERT) (11,12). Whereas large segmental alterations are common in NB, the somatic acquisition of small genetic alterations are relatively rare in sporadic NB, with recurrent alterations mainly found in ALK, ATRX, PTPN11 andTIAM1 (13-15). However, as the use of large-scale, high-throughput techniques, such as next generation sequencing, is expanding, additional recurrent aberrations that contribute to the development and progression of NB are expected to be detected (16).
Recent whole genome sequencing (WGS) studies performed by us and others have detected recurrent structural variants (SVs) that affect the limbic system-associated membrane protein (LSAMP) gene located in chromosomal region 3q13.31 (12,17). LSAMP encodes a protein member of the Ig-like cell adhesion (IgLON) family of proteins, which also include opioid-binding protein/cell adhesion molecule like (OPCML), neuronal growth regulator 1 (NEGR1), neurotrimin (NTM) and IGLON5 (18). All of the IgLON members have been shown to be expressed during central nervous system development, and are involved in cell adhesion, neurite outgrowth (19), dendritic arborization, neuronal development (20) and synapse formation (21,22). Alterations in IgLONs have been reported to be associated with mental disorders (23-25) and also different types of cancer, where they function as tumor suppressor genes (26-28).
The presence of LSAMP has been observed in neural crest cells in experiments conducted in chickens (19), indicating that it may be relevant in the context of NB, as it is considered a malignancy caused by dysregulation of embryonic neural crest development (29). In addition, LSAMP is reportedly involved in axon guidance during the limbic system development (30), neurite outgrowth in dorsal root ganglia (31) and psychiatric disorders (24,32). Notably, it has also previously been reported as a tumor suppressor gene in several types of cancer (33-44).
Given that LSAMP has been shown to have a role in neural development and has been implicated as a tumor suppressor gene in different types of cancer, the present study hypothesized that LSAMP could have a tumor-suppressing capacity in NB. This was investigated in the present study by examining the frequency and extent of recurrent genomic alterations targeting the LSAMP gene in NB tumors and NB cell lines through WGS and single nucleotide polymorphism (SNP)-microarrays, together with in vitro exploration of the functional implication of LSAMP re-expression or silencing.
Materials and methods
Tumor material and cell lines
All NB samples from Swedish patients (n=35) were collected after obtaining written informed consent from their parents or guardians, and were analyzed according to permits approved by the Karolinska Institutet and the Karolinska University Hospital ethics committees (Stockholm, Sweden; approval no. 2009/1369-31/1) in agreement with The Declaration of Helsinki. Sampling was performed during routine clinical procedures, and treatment was performed according to established national and international protocols. Demographic and genomic data of patients are outlined in Table I. The NB cell lines NB1, NB69, SK-N-AS, SK-N-BE(2), SK-N-F1, SK-N-DZ, SK-N-SH, SH-SY5Y, KELLY and IMR-32 were obtained from the American Type Culture Collection. LS and NGP cell lines were kindly provided by Prof. Manfred Schwab (DKFZ, Heidelberg, Germany), whereas the CLB-GAR, CLB-BAR, CLB-PE and CLB-GE cell lines were retrieved from the Centre Léon Bérard (Lyon, France) under a material transfer agreement. All cell lines were cultured in high-glucose DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in an atmosphere containing 5% CO2, and were routinely verified to be negative for mycoplasma, bacterial and fungal infection.
WGS analysis
DNA from primary tumor and corresponding normal samples from 35 patients were analyzed by WGS, including seven patients from our recent study where relapsed tumor material was also subject to sequencing (12). Tumor DNA was extracted from fresh frozen tissue and constitutional DNA from the blood using DNeasy Blood and Tissue Kit (Qiagen GmbH) with sequencing, subsequent bioinformatics handling and variant filtering performed as described previously (12). Briefly, pair-end sequencing was conducted on NovaSeq 6000 (Illumina, Inc.) with an average coverage of at least 60X for tumor material and 30X for constitutional DNA. The Sentieon TNscope software version 201911 (Sentieon, Inc., Mountain View, CA) was used for mapping to hg19, removal of read duplicates, realignment around InDels and variant calling, whereas QIAGEN Clinical Insight Interpret software (version 8.1.20210827; Qiagen GmbH) was used for systematic filtering of called variants. Copy number profiles were generated using the CANVAS tool (version 1.38.0.1554) (45) and somatic SVs were called using the Manta tool (version 1.1.1) (46). All called SVs were evaluated by manual inspection using Integrative Genomics Viewer (version 2.3.81) (47).
SNP-array analysis
CytoScan™ HD-microarrays (cat. no. 901835; Applied Biosystems; Thermo Fisher Scientific, Inc.) were also used to analyze copy number changes on the tumor samples from the 35 patients with NB in combination with 16 NB cell lines [NB1, NB69, CLB-GAR, CLB-BAR, CLB-PE, CLB-GE, SK-N-AS, SK-N-BE(2), SK-N-F1, SK-N-DZ, SK-N-SH, SH-SY5Y, KELLY, IMR-32, LS and NGP] to corroborate and further explore the 3q13.31 chromosomal region. The arrays were scanned using a confocal laser scanner, GeneChip Scanner 3000 (Applied Biosystems; Thermo Fisher Scientific, Inc.) with normalization of the tumor samples performed in silico with genomic annotation based on the hg19 human genome. The procedure for handling CytoScan HD microarrays has been described previously (48).
Sanger sequencing analysis
Primers for LSAMP breakpoint verification were designed either using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) or placed manually with specificity evaluated through UCSC In-Silico PCR (https://genome.ucsc.edu/), and were used together with PLATINUM SuperFI PCR Master Mix (Thermo Fisher Scientific, Inc.) for PCR amplification, according to the manufacturer's instructions. Primer sequences are presented in Table SI. Sanger sequencing of amplicons was performed by Eurofins Genomics following standard conditions. The sequencing visualization and analysis was performed using SnapGene viewer software version 5.1 (www.snapgene.com), to verify LSAMP rearrangements.
Cell transfection, proliferation, viability and differentiation assays
To evaluate the role of LSAMP in proliferation and viability, LSAMP was silenced in the LSAMP-expressing cell lines SH-SY5Y and SK-N-BE(2), whereas re-/overexpression of LSAMP was conducted in the KELLY and SK-N-AS cell lines (with homozygous and heterozygous LSAMP deletion, respectively). Knockdown of LSAMP was performed using pre-designed short hairpin (sh)RNA lentiviral particles from Santa Cruz Biotechnology, Inc. LSAMP shRNA (h) Lentiviral Particles (cat. no. sc-78206-V) were used and Control shRNA Lentiviral Particles-A (cat. no. sc-108080) were used as the control, whereas copGFP-Control Lentiviral Particles (cat. no. sc-108084) was used for optimization and determination of initial transfection efficiency in parallel experiments. Transduction using lentiviral particles was performed with 1×105 particles and 1×105 cells, corresponding to a multiplicity of infection of 1, in 1 ml medium per well in 12-well plates using Polybrene (cat. no. sc-134220) at a final concentration of 8 µg/ml. After 48 h transduction, the medium containing lentiviral particles and Polybrene was replaced with complete medium lacking Polybrene. Stable cells were selected by adding puromycin (2 µg/ml) to the medium 72 h post-transduction, with changes of fresh medium supplemented with puromycin (2 µg/ml) every 3-4 days during the expansion for further analysis. To re-/overexpress LSAMP, pCMV6-AC-LSAMP and pCMV6-AC (control) plasmids (GeneArt; Thermo Fisher Scientific, Inc.) were used. The GFP-tagged constructs pCMV6-AC-LSAMP-GFP and pCMV6-AC-GFP were seeded in parallel experiments in order to determine initial transfection efficiency as well as cellular protein localization. All constructs were verified by Sanger sequencing. The transfection was conducted in 10-cm plates containing 2.5×106 cells/well using 1 µg respective vector dissolved in Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). The medium was changed 24 h after transfection and 200 µg/ml neomycin was added 48 h post-transfection for the selection of stable transfects. The medium was replaced with fresh medium supplemented with neomycin (200 µg/ml) every 3-4 days and cells were expanded for further analyses. Real-time measurements of cell proliferation were performed for 96 h using E-plates in the xCELLigence RTCA DP Instrument (Agilent Technologies, Inc.) according to the manufacturer's protocol, which uses impedance for live monitoring of proliferation. By contrast, cell viability was monitored after 72 h of seeding using Presto Blue HS (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol with a 15-min incubation with the Presto Blue HS reagent prior to absorbance measurement. Each group was assessed four times in 96-well plates and all experiments were repeated three times.
To evaluate the effect of LSAMP silencing on differentiation capacity, SK-N-BE(2) and SH-SY-5Y cells were seeded at a density of 750 cells/well in 96-well plates 1 day before replacing the medium with differentiation medium, which consisted of high-glucose medium supplemented with 2.5% FBS and 7.5 µM retinoic acid (RA; MilliporeSigma). High-glucose medium supplemented with 2.5% FBS and 0.075% EtOH was used as a control. The medium was then changed 4 days after seeding and neurite extension was evaluated using a fluorescence microscope after 7 days of RA incubation using a neurite outgrowth staining kit (cat. no. A15001; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.
Reverse transcription-quantitative PCR (RT-qPCR)
RNA was extracted from 14 NB cell lines and transfected cells (maximum 80% confluence) using RNeasy plus mini kit (Qiagen GmbH), and was quantified spectrophotometri cally using a NanoDrop spectrophotometer (NanoDrop; Thermo Fisher Scientific, Inc.). cDNA synthesis was conducted using the Superscript IV VILO master mix according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.). qPCR was performed using TaqMan™ Fast Advanced Master Mix (Applied Biosystems; Thermo Fisher Scientific) under standard conditions in a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific) under the following conditions: Initial hold at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 3 sec and annealing/extension at 60°C for 30 sec. The following probes were used: LSAMP pre-designed TaqMan probes covering three different exon boundaries (Exon 1-2; Hs01082649_m1, exon 2-3; Hs01082650_m1 and exon 4-5; Hs01082652_m1; Thermo Fisher Scientific, Inc.) and two housekeeping gene probes, qA-01-0104P5 (ACTB) and qA-01-0105P5 (YWHAC) (both from TATAA Biocenter AB). The housekeeping genes used, ACTB and YWHAC, were selected and verified using a human reference gene panel (cat. no. A101) from TATAA Biocenter AB and analyzed by GeneEx (https://bio.tools/geneex). Gene expression analysis was performed using the 2−ΔΔCq method (49).
Kaplan-Meier analysis
Kaplan-Meier analysis with calculation of log-rank test for overall and event-free survival probability in relation to the expression levels of LSAMP and other members of the IgLON family (OPCML, NTM, IGLON5 and NEGR1) was performed using the Kaplan-scan cutoff method in 'R2: Genomic Analysis and Visualization Platform' (http://r2.amc.nl) with the publicly available dataset 'Tumor Neuroblastoma-SEQC-498-rpm-Seqnb1' (n=498). The Kaplan-scan cutoff method examines every increasing expression value as cutoff for log-rank test in order to find the optimal segregation point of two groups based on gene expression. This method then presents the most statistically significant cutoff with corresponding Bonferroni corrected P-value together with the initial non-corrected P-value.
Statistical analysis
The proliferation and viability studies were performed on at least three repeats per respective cell line, and included at least four replicates per sample and assay. Cell proliferation was measured every hour during 96 h. Two-way repeated measures (RM) ANOVA was used to determine the significance of the proliferation differences between two groups over time. The Geisser-Greenhouse ε correction was applied following ANOVA, since the experiment includes multiple measurements over time, the sphericity of the sample cannot be assumed. Cell viability was measured 72 h after cell seeding. Unpaired two-sided t-test analysis was performed using the normalized mean value of each assay, to compare the means of two groups, assuming Gaussian distribution. LSAMP expression levels in the different NB cell lines were quantified by RT-qPCR in triplicate and qPCR was repeated three times. Differences in LSAMP expression in cell lines were analyzed using unpaired two-sided t-test, comparing the means from two groups with different LSAMP status (wild type vs. SVs). Comparison of LSAMP expression levels in human NB tumors with or without MYCN amplification was performed by one-way ANOVA, whereas the correlation of gene expression levels of LSAMP, OPCML, NTM, IGLON5 and NEGR1 was assessed using Pearson correlation coefficient analysis, both using the 'Tumor Neuroblastoma-SEQC-498-rpm-Seqnb1' (n=498) dataset (http://r2.amc.nl). All statistical analyses were performed using GraphPad 9.2.0 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.
Results
Copy number analysis detects recurrent focal alterations of LSAMP
Through copy number profiling and structural variation calling from WGS analysis, somatic focal rearrangements located within chromosomal region 3q13.31 were observed at the heterozygous level in tumor tissue from six out of the 35 investigated NB tumor samples (Fig. 1A and B). Analysis of these rearrangements revealed that the breaks were located within or in the vicinity of the LSAMP gene (Table II), resulting in disruption of the gene. SNP-microarray performed on the same tumor tissue verified these segmental alterations (Fig. S1A and B), whereas the specific breakpoints in LSAMP were verified by PCR and Sanger sequencing (Fig. S2).
From the seven tumors included in our previous study, from which primary and relapsed material was available, LSAMP rearrangement was present in two. In these two cases, the rearrangements were present in the relapsed tumors only and not in the primary tumors (cases NBP26 and NBP28) (10) (Fig. 1B; left panel). WGS did not detect any non-synonymous mutations in LSAMP among the investigated tumors.
Other large recurrent segmental or genomic alterations detected in the six NB samples with LSAMP rearrangement included 1p/1q-imbalance (n=4), 2p-gain (n=5), 3p-deletion (n=4), 4p-deletion (n=2), 17q-gain (n=6), 11q-deletion (n=3), MYCN-amplification (n=2), and break in proximity of TERT or in ATRX (n=4) (Fig. 1A; Table II). Major genomic alterations detected in the full cohort are summarized in Table I. In addition to the primary tumor material, 16 NB cell lines were analyzed by SNP-microarrays. This analysis revealed that seven out of the 16 cell lines (NB1, CLB-GE, CLB-PE, CLB-GAR, SK-N-AS, KELLY and NGP) had segmental alterations involvingLSAMP, including a homozygous deletion within LSAMP detected in KELLY (Fig. 1B, right panel; Fig. S1C).
Segmental alterations are associated with a decreased gene expression in NB cell lines
In order to analyze whether the expression levels of LSAMP differed between the cell lines with and without segmental alterations, TaqMan probes intersecting three different exon boundaries (exons 1-2, 2-3 and 4-5) were utilized (Fig. S3C). After unpaired two-sided t-test, no statistically significant differences in LSAMP expression levels were observed when comparing cell lines with and without 3q13.31 SVs (Figs. 1C and S3A); however, a trend of lower expression was observed in the cell lines with 3q13.31 SVs. Furthermore, in the case of a homozygous deletion break point inside of LSAMP, i.e. in the KELLY cell line, no expression was detected at exons 1-2 and 2-3, and very low expression was detected at exon 4-5 (Fig. S3A).
Knockdown of LSAMP restimulates NB cell proliferation in vitro
To investigate the effect of LSAMP knockdown on the LSAMP-expressing cell lines SH-SY5Y and SK-N-BE(2), shRNA lentiviral particles were used. Initial transduction success for SH-SY5Y and SK-N-BE(2) cells was ~70% for both cell lines, as judged by the number of GPF-expressing cells among control cells transduced with copGFP-Control lentiviral particles (data not shown). Determination of knockdown efficiency through qPCR showed a ~50% reduction in LSAMP expression levels in silenced cells as compared with stable cells transduced with control shRNA (Fig. S3B, left panel). After the selection of stable clones, proliferation and viability analyses were conducted. Knockdown of LSAMP led to statistically significant increase in proliferation in both SH-SY5Y and SK-N-BE(2) cell lines after 96 h [two-way RM ANOVA with the Geisser-Greenhouse ε correction; SH-SY5Y, P<0.0001; SK-N-BE(2), P=0.045; Fig. 2A]. A significant difference in viability after 72 h was also seen after LSAMP knockdown in SH-SY5Y (unpaired t-test; P=0.004) and SK-N-BE(2) (unpaired t-test; P=0.0004) cells (Fig. 2C). Knockdown of LSAMP, with or without the presence of RA, did not exhibit any major effect on neurite extension in SH-SY5Y and SK-N-BE(2) cells (Fig. S4).
LSAMP re- and overexpression inhibits NB proliferation and viability in vitro
In order to investigate how the re-expression and overexpression of LSAMP can affect cell proliferation and viability, KELLY cells (in which LSAMP is homozygously deleted) and SK-N-AS cells (which have a heterozygous LSAMP deletion) were used, as both display low expression levels of LSAMP. Initial transfection efficacy for KELLY and SK-N-AS cells was 70%, as judged by the expression of GFP in cells transfected with pCMV6-AC-GFP at the same occasion (data not shown). Evaluation by qPCR showed a significant increase of LSAMP expression levels in LSAMP-transfected cells as compared with in pCMV6-AC-tranfected controls (Fig. S3B, right panel). Re- and overexpression of LSAMP in KELLY and SK-N-AS cells both led to a significant decrease in cell proliferation after 96 h (two-way RM ANOVA with Geisser-Greenhouse's ε correction; P<0.0001; Fig. 2B) as well as reduced viability after 72 h in KELLY (unpaired t-test; P=0.0022) and SK-N-AS (unpaired t-test; P=0.0032) cells (Fig. 2D). Regarding KELLY cells, viability was also significantly reduced after 96 h (P<0.001; Fig. S3D). Moreover, preliminary data of LSAMP overexpression in SH-SY5Y cells also exhibited a significant reduction in viability (data not shown).
Low expression of LSAMP and other members of the IgLON family in patients with NB is associated with poor survival
To investigate the prognostic value of LSAMP in NB, the expression levels of LSAMP in a publicly available and validated cohort of 498 NB tumors were studied. In this dataset, low LSAMP expression was significantly associated with poor patient overall survival and event-free survival (Fig. 3). The association ofLSAMP with poor overall and event-free survival was most prominent among the non-MYCN-amplified cases and to lesser extent among the MYCN-amplified cases (Fig. S5A), although no statistically significant difference in expression was seen when comparing MYCN-amplified vs. non-MYCN-amplified NB (Fig. S5B and C). Similar results were seen for other NB cohorts also publicly available from the R2 genomic analysis and visualization platform (data not shown).
Investigation of other members of the IgLON family, OPCML, NTM, IGLON5 and NEGR1, using the same NB dataset as aforementioned, revealed that low expression of these genes was also significantly associated with poor overall and event-free survival (Fig. S6A). However, no strong correlation in gene expression was detected between any of the different IgLON members (Fig. S6B).
Discussion
By WGS and SNP-microarrays, recurrent focal segmental rearrange ments affecting the LSAMP gene located at chromosomal region 3q13.31 were de tected in six out of 35 NB tumor samples (17%) as well as in seven out of 16 NB cell lines (44%). The LSAMP alterations were co-occurring with several other genomic alterations, including MYCN-amplification, 1p-deletion, 11q-deletion and 17q-gain, in which the latter was observed in all cases with LSAMP aberration. The co-occurrence of 17q-gain indicated that they are a part of a subgroup associated with a worse prognosis. However, segmental and numerical gains affecting the long arm of chromosome 17 is the most common chromosomal aberration in NB present in all genomic subgroups (7), and thus not unique for cases with LSAMP alterations. WGS did not detect any pathogenic or likely pathogenic variants in LSAMP. However, the genetic etiology with few somatic mutations indicated that NB is mainly driven by genomic events that cause copy number alterations, lead to enhancer hijacking or disruption of genes. Thus, the lack of point mutations in favor of segmental alterations of LSAMP is not completely surprising.
LSAMP has a selective role in neuronal growth, which is also interesting from a NB research perspective. In addition, recurrent alterations in other genes associated with neuro-development and NB have recently been described by Lopez et al (17). Notably, LSAMP has previously been described as a tumor suppressor gene in osteosarcoma (33-37), ovarian cancer (38), renal cell carcinoma (39), prostatic cancer (40,41), epithelioid glioblastoma (42), myeloid leukemia (43) and recently in lung cancer (44). Gene expression analysis among NB cell lines indicated that the average expression levels were lower in LSAMP-rearranged samples, although no statistically significant difference was detected between the groups; however, in the cell line KELLY with a homozygous deletion rearrangement inside LSAMP, no expression levels were detected at exons 1-2 and 2-4. The low expression of LSAMP in cell lines lacking the 3q13.31 rearrangement could be due to epigenetic alterations or other mechanisms, as found in lung cancer and clear cell renal cell carcinoma (33,38); however, further studies are needed to clarify this. In order to study the expression and regulation of LSAMP in NB, knockdown and re-/overexpression experiments were performed in different NB cell lines. LSAMP knockdown led to a significant increase in cell proliferation and viability in the LSAMP wild-type cell lines SH-SY5Y and SK-N-BE(2). These results are in concordance with previous publications (33-44), suggesting that LSAMP could be a putative tumor suppressor gene also in NB. In addition, re-expression of LSAMP in KELLY cells (with homozygous LSAMP deletion) and overexpression of LSAMP in SK-N-AS cells (with heterozygous LSAMP deletion) inhibited cell proliferation and viability. The difference in the level of effect on proliferation between SK-N-AS cells, which had major cell loss, compared with KELLY cells, which had only a decrease in proliferation, could possibly be due to the hetero- and homozygous status of LSAMP deletion in the respective cell line. As evaluation of apoptosis was not performed, the current study is restricted as we cannot distinguish between cell death or decreased proliferation as a cause of decreased cell viability.
The present study also revealed that low expression of LSAMP was associated with poor overall survival in patients with NB, which supports a possible role in aggressive tumor behavior. This association was most evident for the non-MYCN-amplified tumors. In the present cohort of mainly high-risk cases, SVs affecting LSAMP were present in different genomic subgroups (MYCN-amplified, MYCN-amplified together with 11q-deletion, 11q-deleted, 17q-gain and other structural aberrations). A study of a larger cohort reported that SVs affecting LSAMP were predominantly found in non-MYCN-amplified high-risk tumors (17). However, the higher occurrence of LSAMP SVs in cell lines relative to patient tumor samples indicated that this alteration might be a later event contributing to tumor progression rather than initiation. Furthermore, for two of the tumors, LSAMP rearrangements were detected in the tumor during relapse, but not at the time of diagnosis. These findings also supported the hypothesis that LSAMP alterations are not associated with initial tumor development but rather contribute to the progression of the disease or the promotion of cell migration. No major difference in neurite extension was seen after LSAMP knockdown in NB cell lines. However, the current study is limited and additional investigations exploring longer or other differentiation conditions, together with additional cell lines, is required for a better understanding of LSAMP in the context of cell differentiation.
LSAMP is a member of the IgLON family, which is a subgroup of cell adhesion molecules involved in diverse roles in neuronal development (18), and which can also function as tumor suppressor genes (26,50-53). The main members of this family are all located in other chromosomal regions that are frequently deleted in NB and associated with poor prognosis; NEGR1 is located in 1p31, IGLON5 is located in 19q13, and OPCML and NTM are located in 11q25, a chromosomal region that is commonly in cluded in 11q-deleted NB and also in a subset of MYCN-amplified NB (7,16,54,55). Notably, low expression of these family members was associated with poor overall and event-free survival, but did not exhibit obvious correlation with LSAMP expression levels in NB. In addition, studies in lung cancer have shown that NEGR1 is involved in the regulation of LSAMP and that LSAMP can be linked to cancer cell migration through epithelial-mesenchymal transition (44). A similar finding was reported in another study in which depletion of NEGR1 led to increased cell migration and invasion (53). The presence of LSAMP alterations were detected at the heterozygous level in most cases, which would imply haploinsufficiency; however, an additional level of complexity is added from the fact that IgLONs have a conserved interaction mode with the capability of binding to each other as homodimers and heterodimers (56). LSAMP can heterodimerize with either OPCML or NTM, both 11q-localized (57), and if the dimerization partner of LSAMP is not present, this could also possibly affect proper function. This suggests that decreased levels of LSAMP, possibly in combination with decreased levels of other IgLON members, may facilitate tumorigenesis through mechanisms affecting proliferation, migration or contact inhibition. However, the specific role of the different IgLON family members in NB pathogenesis is so far speculative and requires further investigation. Furthermore, having in consideration the highly conserved molecular features of IgLONs, it would be interesting to study the possible implications of LSAMP downregulation on MAPK/PI3K signaling, as NEGR1 has been reported to be related with this relevant pathway in cancer (18,58).
In conclusion, the present sequencing analyses detected novel recurrent somatic alterations involving LSAMP, a cell adhesion molecule that functions in neural crest development and neurite extension. The in vitro studies revealed that LSAMP may have an anti-proliferative function in NB, possibly through the dysfunction of intercellular adhesion and neural differentiation.
Supplementary Data
Availability of data and materials
The genetic datasets from patients associated with this manuscript are not publicly available due to protection from complete disclosure of genome data according to the consent form. SNP-microarray data generated and/or analyzed during the current study are available in the Gene Expression Omnibus database under accession no. GSE209728 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE209728). Other data-sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
AMM conceived and designed experiments. AMM, AD, JG and SF performed the experiments, analyzed and interpreted the data. PK helped with patient care and obtaining clinical information. SF designed the study, formulated strategy and directed the research. AMM and SF confirmed the authenticity of all the raw data and drafted the manuscript. All authors read and approved final version of the manuscript.
Ethics approval and consent to participate
All NB samples from Swedish patients were collected after obtaining written informed consent from their parents or guardians, and were analyzed according to permits approved by the Karolinska Institutet and the Karolinska University Hospital ethics committees (Stockholm, Sweden; approval no. 2009/1369-31/1) in agreement with The Declaration of Helsinki.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Acknowledgments
Not applicable.
Funding
This work has been supported by grants from the Assar Gabrielsson foundation, the Swedish Childhood Cancer Foundation (grant no. SF: PR2018-099, 15-0061) and the Swedish Research Council (grant no. SF: 521-2014-3031).
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