Investigating Trk receptor signalling in neuroblastoma

Neuroblastoma is a complex paediatric cancer characterised by significant clinical heterogeneity, ranging from spontaneous tumour regression to aggressive metastatic disease. Low-risk patients achieve over 90% survival with minimal treatment, while high-risk patients face poor outcomes, with survival rates below 50%, despite intensive therapies. MYCN is a key oncogenic driver, amplified in half of high-risk cases. Differential expression of the Trk receptors (TrkA, TrkB, TrkC) is also linked to prognosis and divergent clinical phenotypes. TrkA is associated with neuronal differentiation, spontaneous regression, and a favourable prognosis, whereas TrkB correlates with aggressive, high-risk neuroblastomas and poor outcomes. The role of TrkC is less characterised, though it appears similar to TrkA, associated with low-stage disease and better prognosis. The precise molecular mechanisms by which these receptors influence cell fate decisions remain unclear. This study aimed to enhance our understanding of Trk receptor signalling in neuroblastoma and the signalling differences driving distinct cellular phenotypes. We first established a panel of neuroblastoma cell lines with varying MYCN levels and overexpression of TrkA, TrkB, or TrkC. Using a network-biology approach that integrated quantitative mass spectrometry-based proteomics, we reconstructed the temporal TrkA, TrkB, and TrkC signal transduction networks, revealing key differences in intracellular signalling linked to opposing cell fate decisions. Focusing on TrkA and TrkB signalling—which drive neuronal differentiation and proliferation, respectively—we identified p38-MAPK signalling dynamics as a critical mediator of these cell fate decisions. Inhibiting p38 blocked neuronal differentiation, while inhibiting negative regulators of p38 (DUSP10/16) in TrkB-expressing cells promoted differentiation, effectively interconverting cell fate outcomes. Analysis of the TrkA signalling network suggested crosstalk with the CDC42/RhoA/PAK pathway, mediated by interaction with PAK2. These findings highlight the pivotal role of p38-MAPK signalling and its regulatory networks in determining neuroblastoma cell fate, offering potential targets for therapeutic intervention to manipulate differentiation and proliferation pathways in TrkB-expressing neuroblastoma. Investigating the interplay between MYCN status and TrkC signalling, we found that TrkC activation led to neuronal differentiation in MYCN non-amplified cells but promoted proliferation in MYCN-high cells. Temporal phosphoproteomics revealed differential activation of the PKA pathway, crucial for TrkC-mediated differentiation. Manipulating the PKA pathway altered cell fate outcomes, reinforcing its importance. In MYCN-amplified cells, MYCN knockdown increased PKA and CREB activity, shifting the phenotype towards differentiation. Analysis of patient data indicated lower expression of PKA pathway genes in MYCN-amplified tumours, with miR-221—upregulated by MYCN—identified as a suppressor of the PKA/CREB pathway. These findings suggest that targeting the PKA pathway could be a promising therapeutic strategy for inducing differentiation in high-risk MYCN-amplified neuroblastoma. To further validate these findings, we developed preclinical zebrafish models with TrkB or TrkC overexpression in the sympathetic nervous system, providing a physiologically relevant model to identify potential therapeutic targets. Overall, this study offers valuable insights into Trk receptor signalling in neuroblastoma and its crucial role in mediating cell fate decisions. We demonstrate the potential to manipulate these decisions, indicating that targeting the intracellular signalling network to guide cells toward a more differentiated and favourable phenotype could represent a promising therapeutic strategy.