Role of an N2-Src-COPII pathway in neuroblastoma differentiation

Neuroblastoma is the most common extracranial solid tumour in childhood and nearly half of neuroblastoma cases occur in children less than two years of age. This cancer is thought to arise due to an alteration during neuronal differentiation and only 30 % of patients with high-risk tumours survive. High-risk neuroblastoma cases are treated with retinoic acid to induce differentiation of the tumour cells left after tumour resection, but these tumours are usually refractive to retinoic acid and other post-surgery therapies. Intriguingly, a positive outcome for low risk disease correlates with high expression levels in the tumour of a neuronal isoform of the ubiquitously expressed C-Src kinase, N2-Src kinase, which is thought to induce spontaneous differentiation of the neuroblastoma to a harmless neuronal phenotype. Indeed, preliminary experiments in our laboratory have shown that overexpression of N2-Src in retinoic acid-resistant neuroblastoma cells is sufficient to differentiate them. It was also shown that N2-Src kinase interacts with proteins of the COPII complex, which coats secretory vesicles that traffic from the ER to the Golgi apparatus, and, interestingly, a preliminary finding showed that the overexpression of the COPII protein Sec23A disrupts N2-Src-dependent differentiation. In this study, I investigated the interplay between N2-Src and the COPII complex in an inducible N2-Src expressing cell line. The aims of my research were to i) understand how N2-Src regulates COPII transport and to ii) design and validate a tool that can be used to understand whether the N2-Src dependent effects on cell morphology and migration require the COPII complex. I observed the trend of N2-Src decreasing migration, both in a random migration assay and in a wound healing assay. Moreover, an assay for COPII transport revealed a trend of N2-Src increasing the number of secretory vesicles. Furthermore, these vesicles were eventually redistributed along neurite-like processes that the cells extend when they express the kinase. I also designed and validated some shRNA constructs able to silence the protein expression of the COPII coat protein Sec23A, which could be used to verify if the silencing of this protein increases N2-Src-dependent differentiation. Taken together, these data lead to the conclusion that N2-Src might regulate COPII transport. Future differentiation therapies for neuroblastoma could be targeted to the N2-Src/COPII pathway.