The Role of LIN28B Oncogene in Regulating Migration & Invasion of Neuroblastoma Cells

Corallo Diana1, Donadon Michael1, Pantile Marcella1, Daniele Boso1, Ori Michela2, Frasson Chiara3, Zanon Carlo3, Basso Giuseppe4, Tonini Gian Paolo1 and Aveic Sanja1,5 1. Neuroblastoma Laboratory, Fondazione Istituto di Ricerca Pediatrica Città della Speranza, Padua, Italy. 2. Unit of Cell and Developmental Biology, Department of Biology, University of Pisa, Italy. 3. Fondazione Istituto di Ricerca Pediatrica Città della Speranza, Padua, Italy. 4. Department of Women and Child Health, Haematology-Oncology Clinic, University of Padua, Italy. 5. Department of Dental Materials and Biomaterials Research, RWTH Aachen University Hospital, Aachen, Germany.

The RNA-binding protein LIN28B coordinates developmental timing and stem cell identity by suppressing the Let-7 family of microRNAs. The ectopic expression of LIN28B is associated with cell transformation and tumor onset. In patients affected by neuroblastoma, a pediatric solid tumor deriving from neural crest cells (NCC), dysregulated LIN28B expression determines poor prognosis and metastasis occurrence. However, how LIN28B sustains neuroblastoma cell dissemination is still under investigation and the comprehension of the molecular mechanism supporting this pro-metastatic phenotype is the main objective of this study.

The two vertebrate models, Danio rerio and Xenopus leavis, have been exploited for a guided, transient LIN28B overexpression in NCC. The NCC migration was examined in vivo by the 3D time-lapse and live-cell imaging. As the in vitro model with ectopic LIN28B expression, a doxycycline-inducible neuroblastoma SH-SY5Y LIN28B cell line has been generated. Changes in the cell morphology or molecular phenotype were analyzed by the in situ hybridization, immunolabeling, flow cytometry, and transcriptomic analyses. Lin28B-dependent cell migration pattern and their invasive capabilities have been analyzed in 2D and 3D in vitro conditions and validated adopting in vivo xenotransplantation zebrafish model.

In vivo, the forced overexpression of LIN28B impeded differentiation of the sympathoadrenergic cells and significantly decreased the migration of trunk NCC toward the dorsal aorta. In vitro, ectopic expression of LIN28B correlated with the pro-migratory and pro-invasive phenotypes of neuroblastoma cells. This feature was supported by the epithelial-to-mesenchymal transition (EMT) that occurred through the ITGA5 and ITGA6 dependent activation of the Src/PI3K/AKT regulatory axis. The former result was sustained by an increased number of focal-adhesion foci in LIN28B cells. Noteworthy, an altered expression of genes regulating cell interaction with the extracellular matrix (ECM) proteins and involved in the regulation of angiogenesis was detected. The latter finding was confirmed in the 3D conditions when LIN28B neuroblastoma cells were co-cultured with endothelial cells, showing interaction with tubular-like structures generated by endothelial cells or more intense neo-vessels branching in vivo.

Taken together, our results establish the relevance of LIN28B in the regulation of not only NCC stemness but also their migration during embryonic development. In addition, our findings imply the possible involvement of LIN28B oncogene in the regulation of pre-dissemination steps of invasion, namely intravasation and extravasation. Due to this multi-level actions of LIN28B, its ectopic expression is sufficient to cause cell transformation and subsequently sustain their invasive potential required for the formation of metastatic disease. Importantly, the complexity of the LIN28B-dependent network implies the use of advanced molecular biology approaches and engineering platforms allowing a comprehensive examination of the pro-metastatic features of tumor cells.

• Funding: Fondazione Italiana per la Lotta al Neuroblastoma and Fondazione Città della Speranza • Conflict of interest: The authors have no conflicts of interest to declare.