L1-CAM knockdown reduces tumorsphere formation in MycN amplified human neuroblastoma stem-like cell lines

Abstract

Childhood neuroblastoma is one of the most malignant types of cancer leading to a high mortality rate. The proto-oncogene Myelocytomatosis Neuroblastoma (MycN) is known to be amplified in this type of cancer, thus promoting high malignancy and resistance. The L1 Cell Adhesion Molecule (L1-CAM) truncation has been found to be upregulated in many types of malignant cancers. Previous studies have concluded that L1-CAM couples with Receptor Tyrosine Kinases (RTKs) that phosphorylate many pathways, leading to gene transcription, mobility, and inhibition of apoptosis. In the current work, we explored the interplay between L1-CAM and MycN as well as the role played by the receptor tyrosine kinases on tumorigenicity in neuroblastoma cells. We investigated the effect of L1-CAM knock-down and RTKs inhibition on tumorsphere formation and cellular proliferation in the MycN-amplified IMR-32 cell line, compared to the non MycN-amplified SK-N-SH neuroblastoma cells. Tumorsphere formation is one of the indicators of aggressiveness in cancer stem-like cells. Cells were grown in stem cell specific medium enriched with growth factors; this growth condition transformed the IMR-32 cell line from a monolayer of adherent cells into large floating tumorspheres that reached 0.5mm in diameter, compared with the SK-N-SH cell line that did not form any. Tumorsphere formation and cellular proliferation was significantly inhibited after L1-CAM knock-down in the IMR-32 cells. Sunitinib malate (Sutent) was used to determine the effect of RTKs inhibition on cell behavior. Sutent treatment (0.2uM) significantly inhibited tumorsphere formation and cellular proliferation in IMR-32 cells. L1-CAM knock down and Sutent did not show any synergy and the si-RNA treatment alone was more efficient than the drug. Taken together, our data shows the importance of L1-CAM interplay with MycN and RTK in MycN amplified neuroblastoma malignancy and motility.