Elucidating the physiological adaptation of loss of retinoblastoma protein in conjunction with hypoxia in neuroblastoma cells.

Neuroblastoma is a malignancy of multipotent embryonic neural crest cells and is the most common cancer in infancy. Since neuroblastoma tumours originate from immature sympathetic cells called neuroblasts, the effect of hypoxia on these cells is of great significance. Low oxygen pressure (hypoxia) is a physiological condition that facilitates increased malignancy and tumour progression in several solid cancers. Hypoxia Inducible factors (HIF)-1 and HIF-2 and their dimerization partner TRIP230 are the principle transcriptional regulators of the hypoxic response. Our group has previously shown that the tumour suppressor retinoblastoma protein (Rb) acts as a transcriptional repressor of hypoxia by its ability to associate with HIF-1. The role of loss of Rb in facilitating the hypoxia related-genetic programs has yet to be studied in neuroblastoma. We used CRISPR-Cas9 technologies to genetically knock-out Rb expression in two non MYCN amplified neuroblastoma cell lines, SH-SY5Y and SK-N-AS. We used a microarray platform to compare the steady-state expression levels of mRNA from these two cell lines to determine aberrant expression of hypoxia related genetic programs that increase cell motility and invasion and promote metastasis. Using migration assays, qRT-PCR and Western blot analysis we have identified multiple genes whose expression is significantly upregulated with the loss of Rb in conjunction with hypoxia. The identification and characterization of these genes could provide the basis for developing novel therapies and new diagnostic markers for treatment for children with non-MYCN amplified neuroblastoma.