Susanne Fransson

The Risk-Associated Long Noncoding RNA NBAT-1 Controls Neuroblastoma Progression by Regulating Cell Proliferation and Neuronal Differentiation

Neuroblastoma is an embryonal tumor of the sympathetic nervous system and the most common extracranial tumor of childhood. By sequencing transcriptomes of low- and high-risk neuroblastomas, we detected differentially expressed annotated and nonannotated long noncoding RNAs (lncRNAs). We identified a lncRNA neuroblastoma associated transcript-1 (NBAT-1) as a biomarker significantly predicting clinical outcome of neuroblastoma. CpG methylation and a high-risk neuroblastoma associated SNP on chromosome 6p22 functionally contribute to NBAT-1 differential expression. Loss of NBAT-1 increases cellular proliferation and invasion. It controls these processes via epigenetic silencing of target genes. NBAT-1 loss affects neuronal differentiation through activation of the neuronal-specific transcription factor NRSF/REST. Thus, loss of NBAT-1 contributes to aggressive neuroblastoma by increasing proliferation and impairing differentiation of neuronal precursors.

Genetic and epigenetic changes in the common 1p36 deletion in neuroblastoma tumours

Chromosome 1p is frequently deleted in neuroblastoma (NB) tumours. The commonly deleted region has been narrowed down by loss of heterozygosity studies undertaken by different groups. Based on earlier mapping data, we have focused on a region on 1p36 (chr1: 7 765 595–11 019 814) and performed an analysis of 30 genes by exploring features such as epigenetic regulation, that is DNA methylation and histone deacetylation, mutations at the DNA level and mRNA expression. Treatment of NB cell lines with the histone deacetylase inhibitor trichostatin A led to increased gene transcription of four of the 30 genes, ERRFI1 (MIG-6), PIK3CD, RBP7 (CRBPIV) and CASZ1, indicating that these genes could be affected by epigenetic downregulation in NBs. Two patients with nonsynonymous mutations in the PIK3CD gene were detected. One patient harboured three variations in the same exon, and p.R188W. The other patient had the variation p.M655I. In addition, synonymous variations and one variation in an intronic sequence were also found. The mRNA expression of this gene is downregulated in unfavourable, compared to favourable, NBs. One nonsynonymous mutation was also identified in the ERRFI1 gene, p.N343S, and one synonymous. None of the variations above were found in healthy control individuals. In conclusion, of the 30 genes analysed, the PIK3CD gene stands out as one of the most interesting for further studies of NB development and progression.

A cluster of genes located in 1p36 are down-regulated in neuroblastomas with poor prognosis, but not due to CpG island methylation

BackgroundA common feature of neuroblastoma tumours are partial deletions of the short arm of chromosome 1 (1p-deletions). This is indicative of a neuroblastoma tumour suppressor gene being located in the region. Several groups including our have been studying candidate neuroblastoma genes in the region, but no gene/genes have yet been found that fulfil the criteria for being a neuroblastoma tumour suppressor. Since frequent mutations have not been detected, we have now analyzed the expression and promoter CpG island methylation status of the genes UBE4B, KIF1B, PGD, APITD1, DFFA and PEX14 in the 1p36.22 region in order to find an explanation for a possible down-regulation of this region.ResultsThe current study shows that gene transcripts in high stage neuroblastoma tumours are significantly down-regulated compared to those in low stage tumours in the 1p36.22 region. CpG island methylation does not seem to be the mechanism of down-regulation for most of the genes tested, since no methylation was detected in the fragments analyzed. One exception is the CpG island of APITD1. Methylation of this gene is also seen in blood from control individuals and is therefore not believed to participate in tumour development.ConclusionThe genes UBE4B, KIF1B, PGD, APITD1, DFFA and PEX14 are down-regulated in high stage NB tumours, a feature that can not be explained by CpG island methylation.

Neuroblastoma tumors with favorable and unfavorable outcomes: Significant differences in mRNA expression of genes mapped at 1p36.2.

The distal part of 1p is frequently deleted in aggressive neuroblastoma, and the region is believed to harbor one or more tumor suppressor genes relevant to tumor development. To analyze differences among neuroblastoma tumors, an expression profile was established for the genes mapped within a previously described shortest region of overlap of deletions at 1p36.2. The gene expression levels were quantified by TaqMan real‐time (RT)‐PCR for 30 transcripts using 55 primary neuroblastoma tumors. Here we report on a significant decrease in gene expression of the genes RERE, PIK3CD, LZIC, PGD, and PEX14 and an increase of SLC2A5 when comparing tumors of favorable biology to Stage 4 neuroblastomas. When comparing 1p‐deleted tumors of all stages to tumors with an intact 1p, a significant difference at gene‐by‐gene level in TNFRSF9, RERE, PIK3CD, CLSTN1, CTNNBIP1, and CASZ1 was detected. A complete loss of expression could not be seen for any single gene analyzed. Several of the genes with diminished expression in unfavorable or 1p‐deleted tumors have functions that could contribute to tumor development. It is also possible that a combination of lowly expressed genes at 1p, rather than one single classical tumor suppressor gene, causes the unfavorable outcome associated with 1p‐deletion in neuroblastoma.

Intragenic Anaplastic Lymphoma Kinase (ALK) Rearrangements: Translocations as a Novel Mechanism of ALK Activation in Neuroblastoma Tumors

Anaplastic lymphoma kinase (ALK) has been demonstrated to be deregulated in sporadic as well as in familiar cases of neuroblastoma (NB). Whereas ALK‐fusion proteins are common in lymphoma and lung cancer, there are few reports of ALK rearrangements in NB indicating that ALK mainly exerts its oncogenic capacity via activating mutations and/or overexpression in this tumor type. In this study, 332 NB tumors and 13 cell lines were screened by high resolution single nucleotide polymorphism microarray. Gain of 2p was detected in 23% (60/332) of primary tumors and 46% (6/13) of cell lines, while breakpoints at the ALK locus were detected in four primary tumors and two cell lines. These were further analyzed by next generation sequencing and a targeted enrichment approach. Samples with both ALK and MYCN amplification displayed complex genomic rearrangements with multiple breakpoints within the amplicon. None of the translocations characterized in primary NB tumors are likely to result in a chimeric protein. However, immunohistochemical analysis reveals high levels of phosphorylated ALK in these samples despite lack of initial exons, possibly due to alternative transcription initiation sites. Both ALK proteins predicted to arise from such alterations and from the abnormal ALK exon 4–11 deletion observed in the CLB‐BAR cell line show strong activation of downstream targets STAT3 and extracellular signal‐regulated kinase (ERK) when expressed in PC12 cells. Taken together, our data indicate a novel, although rare, mechanism of ALK activation with implications for NB tumorigenesis.

Stage-dependent expression of PI3K/Akt‑pathway genes in neuroblastoma.

The phosphoinositide-3 kinase (PI3K) pathway plays a critical role in cancer cell growth and survival and has also been implicated in the development of the childhood cancer neuroblastoma. In neuroblastoma high mRNA expression of the PI3K catalytic isoform PIK3CD is associated to favorable disease. Yet, activation of Akt is associated with poor prognosis. Since the contribution of the numerous members of this pathway to neuroblastoma pathogenesis is mainly unknown, genes of the PI3K/Akt pathway were analyzed at the mRNA level through microarrays and quantitative real-time RT-PCR (TaqMan) and at the protein level using western blot analysis. Five genes showed lower mRNA expression in aggressive compared to more favorable neuroblastomas (PRKCZ, PRKCB1, EIF4EBP1, PIK3RI and PIK3CD) while the opposite was seen for PDGFRA. Clustering analysis shows that the expression levels of these six genes can predict aggressive disease. At the protein level, p110δ (encoded by PIK3CD) and p85α isomers (encoded by PIK3R1) were more highly expressed in favorable compared to aggressive neuroblastoma. Evaluation of the expression of these PI3K genes can predict aggressive disease, and indicates stage-dependent involvement of PI3K-pathway members in neuroblastoma.

p37δ is a new isoform of PI3K p110δ that increases cell proliferation and is overexpressed in tumors

The phosphatidylinositol 3-kinases (PI3Ks) regulate cell growth, proliferation and survival, and are frequently affected in human cancer. PI3K is composed of a catalytic subunit, p110, and a regulatory subunit, p85. The PI3K catalytic subunit p110δ is encoded by PIK3CD and contains p85- and RAS-binding domains, and a kinase domain. Here we present an alternatively spliced PIK3CD transcript encoding a previously unknown protein, p37δ, and demonstrate that this protein is expressed in human ovarian and colorectal tumors. p37δ retains the p85-binding domain and a fraction of the RAS-binding domain, lacks the catalytic domain, and has a unique carboxyl-terminal region. In contrast to p110δ, which stabilizes p85, p37δ promoted p85 sequestering. Despite the truncated RAS-binding domain, p37δ bound to RAS and we found a strong positive correlation between the protein levels of p37δ and RAS. Overexpressing p37δ, but not p110δ, increased the proliferation and invasive properties of HEK-293 cells and mouse embryonic fibroblasts. Cells overexpressing p37δ showed a quicker phosphorylation response of AKT and ERK1/2 following serum stimulation. Ubiquitous expression of human p37δ in the fruit fly increased body size, DNA content and phosphorylated ERK1/2 levels. Thus, p37δ appears to be a new tumor-specific isoform of p110δ with growth-promoting properties.

Estimation of copy number aberrations: Comparison of exome sequencing data with SNP microarrays identifies homozygous deletions of 19q13.2 and CIC in neuroblastoma

In the pediatric cancer neuroblastoma, analysis of recurrent chromosomal aberrations such as loss of chromosome 1p, 11q, gain of 17q and MYCN amplification are used for patient stratification and subsequent therapy decision making. Different analysis techniques have been used for detection of segmental abnormalities, including fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH)-microarrays and multiplex ligation-dependent probe amplification (MLPA). However, as next-generation sequencing becomes available for clinical use, this technique could also be used for assessment of copy number alterations simultaneously with mutational analysis. In this study we compare genomic profiles generated through exome sequencing data with profiles generated from high resolution Affymetrix single nucleotide polymorphism (SNP) microarrays on 30 neuroblastoma tumors of different stages. Normalized coverage reads for tumors were calculated using Control-FREEC software and visualized through a web based Shiny application, prior to comparison with corresponding SNP-microarray data. The two methods show high-level agreement for breakpoints and copy number of larger segmental aberrations and numerical aneuploidies. However, several smaller gene containing deletions that could not readily be detected through the SNP-microarray analyses were identified through exome profiling, most likely due to difference between spatial distribution of microarray probes and targeted regions of the exome capture. These smaller aberrations included focal ATRX deletion in two tumors and three cases of novel deletions in chromosomal region 19q13.2 causing homozygous loss of multiple genes including the CIC (Capicua) gene. In conclusion, genomic profiles generated from normalized coverage of exome sequencing show concordance with SNP microarray generated genomic profiles. Exome sequencing is therefore a useful diagnostic tool for copy number variant (CNV) detection in neuroblastoma tumors, especially considering the combination with mutational screening. This enables detection of theranostic targets such as ALK and ATRX together with detection of significant segmental aneuploidies, such as 2p-gain, 17q-gain, 11q-deletion as well as MYCN amplification.

Aneuploidy in neuroblastoma tumors is not associated with inactivating point mutations in the STAG2 gene.

BackgroundChromosomal instability is a hallmark of human cancer caused by errors in mitotic control and chromosome segregation. STAG2 encodes a subunit of the cohesion complex that participates in mitotic chromatid separation and was recently found to show low expression and inactivating mutations in Ewing’s sarcoma, melanoma and glioblastoma.In the childhood tumor neuroblastoma (NB) segmental chromosomal alterations are associated with poor prognosis whereas tumors displaying whole chromosome gains and losses have a much better prognosis.MethodAs the genetic contribution to aneuploidy is unknown in NB, we investigated the presence of STAG2 mutations through sequence analysis of all 33 coding exons in 37 primary NB tumors.Results and conclusionAs no STAG2 mutation was detected in this study, we conclude that inactivating mutation of STAG2 is not likely causative to neuroblastoma aneuploidy.

Aggressive neuroblastomas have high p110alpha but low p110delta and p55alpha/p50alpha protein levels compared to low stage neuroblastomas

BACKGROUND The phosphoinositide 3-kinase (PI3K)/Akt pathway is involved in neuroblastoma development where Akt/PKB activation is associated with poor prognosis. PI3K activity subsequently activates Akt/PKB, and as mutations of PI3K are rare in neuroblastoma and high levels of PI3K subunit p110delta is associated with favorable disease with low p-Akt/PKB, the levels of other PI3K subunits could be important for Akt activation. METHODS Protein levels of Type IA PI3K catalytic and regulatory subunits were investigated together with levels of phosphorylated Akt/PKB and the PI3K negative regulator PTEN in primary neuroblastoma tumors. Relation between clinical markers and protein levels were evaluated through t-tests. RESULTS We found high levels of p-Akt/PKB correlating to aggressive disease and p-Akt/PKB (T308) showed inverse correlation to PTEN levels. The regulatory isomers p55alpha/p50alpha showed higher levels in favorable neuroblastoma as compared with aggressive neuroblastoma. The PI3K-subunit p110alpha was found mainly in advanced tumors while p110delta showed higher levels in favorable neuroblastoma. CONCLUSIONS Activation of the PI3K/Akt pathway is seen in neuroblastoma tumors, however the contribution of the different PI3K isoforms is unknown. Here we show that p110alpha is preferentially expressed in aggressive neuroblastomas, with high p-Akt/PKB and p110delta is mainly detected in favorable neuroblastomas, with low p-Akt/PKB. This is an important finding as PI3K-specific inhibitors are suggested for enrollment in treatment of neuroblastoma patients.