P0985 - Serine-Threonine Kinase Receptor Associate Protein (STRAP) Confers Stemness in Neuroblastoma

Background: Serine-threonine kinase receptor associated protein (STRAP) is a scaffolding protein that has been implicated in tumor growth and metastasis. The role of STRAP in neuroblastoma (NB) has not been explored. Stem cell-like cancer cells (SCLCCs) are believed to be responsible for tumor cell self-renewal and cancer recurrence. We sought to determine the role of STRAP in neuroblastoma SCLCCs maintenance.

Methods: CRISPR-Cas9 gene editing was utilized to establish stable genetic knockout of STRAP in the human NB cell line SK-N-AS (AS). pcDNA3 c-Flag empty vector or STRAP overexpressing vector were stably transfected in AS cells. Immunoblotting confirmed STRAP knockout and overexpression. Wild type cells and empty vector transfected cells served as controls for knockout and overexpression cells, respectively. Tumor cell stemness was evaluated with quantitative real-time PCR (qPCR), CD133 expression, and extreme limiting dilution assay and compared between wildtype, knockout, empty vector, and STRAP overexpression cells.

Results: qPCR revealed mRNA abundance for stem cell markers, Oct4 and Nanog, was decreased in knockout, whereas overexpression cells had increased Oct4 and Nanog mRNA compared to respective control cells (Figure 1A). Percentage of CD133 positive cells was decreased in knockout cells but increased in overexpression cells (Figure 1B). Tumorsphere formation was decreased in knockout cells at 50 cells per well (0 ± 0% vs 87 ± 9%, knockout vs wildtype, p=0,001), suggested decreased stemness and increased in STRAP overexpression cells at 1000 cells per well (42 ± 0.5% vs 0 ± 0%, overexpression vs empty vector, p<0.001), suggested increased stemness.

Conclusion: STRAP knockout in neuroblastoma cells led to decreased mRNA abundance of stemness markers, CD133 expression, and tumorsphere formation. STRAP overexpression cells showed opposite effects with significantly increased stemness. These novel findings demonstrate that STRAP functions to maintain neuroblastoma SCLCCs and warrants further investigation as a potential therapeutic target in neuroblastoma.