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

Widespread resetting of DNA methylation in glioblastoma-initiating cells suppresses malignant cellular behavior in a lineage-dependent manner

Epigenetic changes are frequently observed in cancer. However, their role in establishing or sustaining the malignant state has been difficult to determine due to the lack of experimental tools that enable resetting of epigenetic abnormalities. To address this, we applied induced pluripotent stem cell (iPSC) reprogramming techniques to invoke widespread epigenetic resetting of glioblastoma (GBM)-derived neural stem (GNS) cells. GBM iPSCs (GiPSCs) were subsequently redifferentiated to the neural lineage to assess the impact of cancer-specific epigenetic abnormalities on tumorigenicity. GiPSCs and their differentiating derivatives display widespread resetting of common GBM-associated changes, such as DNA hypermethylation of promoter regions of the cell motility regulator TES (testis-derived transcript), the tumor suppressor cyclin-dependent kinase inhibitor 1C (CDKN1C; p57KIP2), and many polycomb-repressive complex 2 (PRC2) target genes (e.g., SFRP2). Surprisingly, despite such global epigenetic reconfiguration, GiPSC-derived neural progenitors remained highly malignant upon xenotransplantation. Only when GiPSCs were directed to nonneural cell types did we observe sustained expression of reactivated tumor suppressors and reduced infiltrative behavior. These data suggest that imposing an epigenome associated with an alternative developmental lineage can suppress malignant behavior. However, in the context of the neural lineage, widespread resetting of GBM-associated epigenetic abnormalities is not sufficient to override the cancer genome

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

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

Verification of genes differentially expressed in neuroblastoma tumours: a study of potential tumour suppressor genes

<p>Abstract</p> <p>Background</p> <p>One of the most striking features of the childhood malignancy neuroblastoma (NB) is its clinical heterogeneity. Although there is a great need for better clinical and biological markers to distinguish between tumours with different severity and to improve treatment, no clear-cut prognostic factors have been found. Also, no major NB tumour suppressor genes have been identified.</p> <p>Methods</p> <p>In this study we performed expression analysis by quantitative real-time PCR (QPCR) on primary NB tumours divided into two groups, of favourable and unfavourable outcome respectively. Candidate genes were selected on basis of lower expression in unfavourable tumour types compared to favourables in our microarray expression analysis. Selected genes were studied in two steps: (1) using TaqMan Low Density Arrays (TLDA) targeting 89 genes on a set of 12 NB tumour samples, and (2) 12 genes were selected from the TLDA analysis for verification using individual TaqMan assays in a new set of 13 NB tumour samples.</p> <p>Results</p> <p>By TLDA analysis, 81 out of 87 genes were found to be significantly differentially expressed between groups, of which 14 have previously been reported as having an altered gene expression in NB. In the second verification round, seven out of 12 transcripts showed significantly lower expression in unfavourable NB tumours, <it>ATBF1</it>, <it>CACNA2D3</it>, <it>CNTNAP2</it>, <it>FUSIP1</it>, <it>GNB1</it>, <it>SLC35E2</it>, and <it>TFAP2B</it>. The gene that showed the highest fold change in the TLDA analysis, <it>POU4F2</it>, was investigated for epigenetic changes (CpG methylation) and mutations in order to explore the cause of the differential expression. Moreover, the fragile site gene <it>CNTNAP2 </it>that showed the largest fold change in verification group 2 was investigated for structural aberrations by copy number analysis. However, the analyses of <it>POU4F2 </it>and <it>CNTNAP2 </it>showed no genetic alterations that could explain a lower expression in unfavourable NB tumours.</p> <p>Conclusion</p> <p>Through two steps of verification, seven transcripts were found to significantly discriminate between favourable and unfavourable NB tumours. Four of the transcripts, <it>CACNA2D3</it>, <it>GNB1</it>, <it>SLC35E2</it>, and <it>TFAP2B</it>, have been observed in previous microarray studies, and are in this study independently verified. Our results suggest these transcripts to be markers of malignancy, which could have a potential usefulness in the clinic.</p

A novel 1p36.2 located gene, APITD1, with tumour-suppressive properties and a putative p53-binding domain, shows low expression in neuroblastoma tumours

Neuroblastoma is characterised by a lack of TP53 mutations and no other tumour suppressor gene consistently inactivated has yet been identified in this childhood cancer form. Characterisation of a new gene, denoted APITD1, in the neuroblastoma tumour suppressor candidate region in chromosome 1p36.22 reveals that APITD1 contains a predicted TFIID-31 domain, representing the TATA box-binding protein-associated factor, TAFII31, which is required for p53-mediated transcription activation. Two different transcripts of this gene were shown to be ubiquitously expressed, one of them with an elevated expression in foetal tissues. Primary neuroblastoma tumours of all different stages showed either very weak or no measurable APITD1 expression, contrary to the level of expression observed in neuroblastoma cell lines. A reduced pattern of expression was also observed in a set of various tumour types. APITD1 was functionally tested by adding APITD1 mRNA to neuroblastoma cells, leading to the cell growth to be reduced up to 90% compared to control cells, suggesting APITD1 to have a role in a cell death pathway. Furthermore, we determined the genomic organisation of APITD1. Automated genomic DNA sequencing of the coding region of the gene as well as the promoter sequence in 44 neuroblastoma tumours did not reveal any loss-of-function mutations, indicating that mutations in APITD1 is not a common abnormality of neuroblastoma tumours. We suggest that low expression of this gene might interfere with the ability for apoptosis through the p53 pathway

Glioblastoma Stem Cells Respond to Differentiation Cues but Fail to Undergo Commitment and Terminal Cell-Cycle Arrest.

Glioblastoma (GBM) is an aggressive brain tumor whose growth is driven by stemcell-like cells. BMP signaling triggers cell-cycle exit and differentiation of GBM stemcells (GSCs) and, therefore, might have therapeutic value. However, the epigenetic mechanisms that accompany differentiation remain poorly defined. It is also unclear whether cell-cycle arrest is terminal. Herewe find only a subset ofGSCcultures exhibit astrocyte differentiation in response to BMP. Although overtly differentiated non-cycling astrocytes are generated, they remain vulnerable to cell-cycle re-entry and fail to appropriately reconfigure DNA methylation patterns. Chromatin accessibility mapping identified loci that failed to alter in response to BMP and these were enriched in SOX transcription factor-binding motifs. SOX transcription factors, therefore, may limit differentiation commitment. A similar propensity for cell-cycle re-entry and de-differentiation was observed in GSC-derived oligodendrocyte-like cells. These findings highlight significant obstacles to BMP-induced differentiation as therapy forGBM

Inhibition of N-linked glycosylation impairs ALK phosphorylation and disrupts pro-survival signaling in neuroblastoma cell lines

<p>Abstract</p> <p>Background</p> <p>The Anaplastic Lymphoma Kinase (ALK) is an orphan receptor tyrosine kinase, which undergoes post-translational N-linked glycosylation. The catalytic domain of ALK was originally identified in the t(2;5) translocation that produces the unglycosylated oncogenic protein NPM-ALK, which occurs in Anaplastic Large Cell Lymphoma (ALCL). Recently, both germline and somatic activating missense mutations of ALK have been identified in neuroblastoma (NB), a pediatric cancer arising from neural crest cells. Moreover, we previously reported that ALK expression is significantly upregulated in advanced/metastatic NB. We hypothesized that ALK function may depend on N-linked glycosylation and that disruption of this post-translational modification would impair ALK activation, regardless the presence of either gene mutations or overexpression.</p> <p>Methods</p> <p>We employed tunicamycin to inhibit N-linked glycosylation. The following ALK-positive NB cell lines were used: SH-SY5Y and KELLY (ALK mutation F1174L), UKF-NB3 (ALK mutation R1275Q) and NB1 (ALK amplification). As a control, we used the NB cell lines LA1-5S and NB5 (no ALK expression), and the ALCL cell line SU-DHL1 (NPM-ALK).</p> <p>Results</p> <p>Tunicamycin treatment of ALK-positive NB cells resulted in a hypoglycosylated ALK band and in decreased amounts of mature full size receptor. Concomitantly, we observed a marked reduction of mature ALK phosphorylation. On the contrary, tunicamycin had no effects on NPM-ALK phosphorylation in SU-DHL1 cells. Moreover, phosphorylation levels of ALK downstream effectors (AKT, ERK1/2, STAT3) were clearly impaired only in ALK mutated/amplified NB cell lines, whereas no significant reduction was observed in both ALK-negative and NPM-ALK-positive cell lines. Furthermore, inhibition of N-linked glycosylation considerably impaired cell viability only of ALK mutated/amplified NB cells. Finally, the cleavage of the Poly-ADP-ribose-polymerase (PARP) suggested that apoptotic pathways may be involved in cell death.</p> <p>Conclusions</p> <p>In this study we showed that inhibition of N-linked glycosylation affects ALK phosphorylation and disrupts downstream pro-survival signaling, indicating that inhibition of this post-translational modification may be a promising therapeutic approach. However, as tunicamycin is not a likely candidate for clinical use other approaches to alter N-linked glycosylation need to be explored. Future studies will assess whether the efficacy in inhibiting ALK activity might be enhanced by the combination of ALK specific small molecule and N-linked glycosylation inhibitors.</p

Transcribed-ultra conserved region expression is associated with outcome in high-risk neuroblastoma

<p>Abstract</p> <p>Background</p> <p>Neuroblastoma is the most common, pediatric, extra-cranial, malignant solid tumor. Despite multimodal therapeutic protocols, outcome for children with a high-risk clinical phenotype remains poor, with long-term survival still less than 40%. Hereby, we evaluated the potential of non-coding RNA expression to predict outcome in high-risk, stage 4 neuroblastoma.</p> <p>Methods</p> <p>We analyzed expression of 481 Ultra Conserved Regions (UCRs) by reverse transcription-quantitative real-time PCR and of 723 microRNAs by microarrays in 34 high-risk, stage 4 neuroblastoma patients.</p> <p>Results</p> <p>First, the comparison of 8 short- versus 12 long-term survivors showed that 54 UCRs were significantly (<it>P </it>< 0.0491) over-expressed in the former group. For 48 Ultra Conserved Region (UCRs) the expression levels above the cut-off values defined by ROC curves were strongly associated with good-outcome (OS: 0.0001 <<it>P </it>< 0.0185, EFS: 0.0001 <<it>P </it>< 0.0491). Then we tested the Transcribed-UCR (T-UCR) threshold risk-prediction model on an independent cohort of 14 patients. The expression profile of 28 T-UCRs was significantly associated to prognosis and at least 15 up-regulated T-UCRs are needed to discriminate (<it>P </it>< 0.0001) short- from long-survivors at the highest sensitivity and specificity (94.12%). We also identified a signature of 13 microRNAs differently expressed between long- and short-surviving patients. The comparative analysis of the two classes of non-coding RNAs disclosed that 9 T-UCRs display their expression level that are inversely correlated with expression of 5 complementary microRNAs of the signature, indicating a negative regulation of T-UCRs by direct interaction with microRNAs. Moreover, 4 microRNAs down-regulated in tumors of long-survivors target 3 genes implicated in neuronal differentiation, that are known to be over-expressed in low-risk tumors.</p> <p>Conclusions</p> <p>Our pilot study suggests that a deregulation of the microRNA/T-UCR network may play an important role in the pathogenesis of neuroblastoma. After further validation on a larger independent set of samples, such findings may be applied as the first T-UCR prognostic signature for high-risk neuroblastoma patients.</p

E4 ligase–specific ubiquitination hubs coordinate DNA double-strand-break repair and apoptosis

Multiple protein ubiquitination events at DNA double-strand breaks (DSBs) regulate damage recognition, signaling and repair. It has remained poorly understood how the repair process of DSBs is coordinated with the apoptotic response. Here, we identified the E4 ubiquitin ligase UFD-2 as a mediator of DNA-damage-induced apoptosis in a genetic screen in Caenorhabditis elegans. We found that, after initiation of homologous recombination by RAD-51, UFD-2 forms foci that contain substrate-processivity factors including the ubiquitin-selective segregase CDC-48 (p97), the deubiquitination enzyme ATX-3 (Ataxin-3) and the proteasome. In the absence of UFD-2, RAD-51 foci persist, and DNA damage-induced apoptosis is prevented. In contrast, UFD-2 foci are retained until recombination intermediates are removed by the Holliday-junction-processing enzymes GEN-1, MUS-81 or XPF-1. Formation of UFD-2 foci also requires proapoptotic CEP-1 (p53) signaling. Our findings establish a central role of UFD-2 in the coordination between the DNA-repair process and the apoptotic response