Abstract
Neuroblastoma is one of the commonest and deadliest solid tumours of childhood, and is thought to result from disrupted differentiation of the developing sympathoadrenergic lineage of the neural crest. Neuroblastoma exhibits intra- and intertumoural heterogeneity, with high risk tumours characterised by poor differentiation, which can be attributable to MYCN-mediated repression of genes involved in neuronal differentiation. MYCN is known to co-operate with oncogenic signalling pathways such as Alk, Akt and MEK/ERK signalling, and, together with c-MYC has been shown to be activated by Wnt signalling in various tissues. However, our previous work demonstrated that Wnt3a/Rspo2 treatment of some neuroblastoma cell lines can, paradoxically, decrease c-MYC and MYCN proteins. This prompted us to define the neuroblastoma-specific Wnt3a/Rspo2-driven transcriptome using RNA sequencing, and characterise the accompanying changes in cell biology. Here we report the identification of ninety Wnt target genes, and show that Wnt signalling is upstream of numerous transcription factors and signalling pathways in neuroblastoma. Using live-cell imaging, we show that Wnt signalling can drive differentiation of SK-N-BE(2)-C and SH-SY5Y cell-lines, but, conversely, proliferation of SK-N-AS cells. We show that cell-lines that differentiate show induction of pro-differentiation BMP4 and EPAS1 proteins, which is not apparent in the SK-N-AS cells. In contrast, SK-N-AS cells show increased CCND1, phosphorylated RB and E2F1 in response to Wnt3a/Rspo2, consistent with their proliferative response, and these proteins are not increased in differentiating lines. By meta-analysis of the expression of our 90 genes in primary tumour gene expression databases, we demonstrate discrete expression patterns of our Wnt genes in patient cohorts with different prognosis. Furthermore our analysis reveals interconnectivity within subsets of our Wnt genes, with one subset comprised of novel putative drivers of neuronal differentiation repressed by MYCN. Assessment of β-catenin immunohistochemistry shows high levels of β-catenin in tumours with better differentiation, further supporting a role for canonical Wnt signalling in neuroblastoma differentiation.
Highlights
Neuroblastoma is responsible for approximately 15% of paediatric cancer deaths, with about 40% of patients considered to be high-risk cases with very poor prognosis [1,2]
Using a T-cell factor/lymphoid Enhancer Binding Factor (TCF/Lef) reporter assay (TOPFLASH), we showed that three LGR5-expressing neuroblastoma cell-lines with different oncogenic drivers, SK-N-BE(2)-C (MNA), SH-SY5Y (ALK mutant) and SK-N-AS (NRAS mutant) displayed highly inducible β-catenin-TCF/Lef-regulated transcription when treated with recombinant Wnt3a and R-Spondin 2 (Rspo2), with a strong requirement for LGR5/Rspo2 apparent for maximal induction, as Wnt3a/Rspo2 induction of TOPFLASH was at least 5-fold greater than with Wnt3a alone
Our previous study showed that several established target genes of canonical Wnt signalling were induced in the neuroblastoma cell lines treated with Wnt3a/Rspo2, including AXIN2 and LEF1, we found that SOX2, MYC and MYCN, which were shown to be Wnt pathway target genes studies in other tissue systems, did not exhibit strong induction
Summary
Neuroblastoma is responsible for approximately 15% of paediatric cancer deaths, with about 40% of patients considered to be high-risk cases with very poor prognosis [1,2]. Neuroblastoma is a biologically and clinically heterogeneous cancer arising from the sympathoadrenergic lineage of the neural crest [3]. The paradigmatic mechanism for disrupted differentiation in neuroblastoma is contingent on amplification of the MYCN proto-oncogene [6], with high levels of MYCN protein leading to direct repression of genes necessary for terminal differentiation in the sympathetic nervous system [7,8]. As well as MYCN amplification (MNA), high risk neuroblastomas have been shown to elevate telomerase reverse transcriptase (TERT) expression through deregulatory genomic rearrangements [9,10]. Kinase pathways are deregulated in neuroblastoma, including activating mutations of ALK [11], increased Akt signalling in stage 3 and 4 neuroblastoma [12], and relapsing neuroblastomas displaying mutations in the Ras-MAPK pathway [13,14]
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