Serine-threonine Kinase Receptor Associate Protein (STRAP) Confers Aggressive Phenotype in Neuroblastoma via Regulation of Focal Adhesion Kinase (FAK) Signaling

Background: Serine-threonine kinase receptor associated protein (STRAP) is a scaffolding protein implicated in tumor formation and maintenance. We have demonstrated that STRAP knockout (KO) decreased the malignant phenotype in neuroblastoma while the overexpression (OE) of STRAP increased neuroblastoma cell proliferation, motility, and anchorage independent growth. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, is regulated by cell-cell interaction and scaffolding proteins. FAK prevents tumor cell anoikis, thereby playing an important role in tumor progression in many cancers including neuroblastoma. Since STRAP is a known scaffolding protein and FAK activation is regulated by other scaffolding proteins, we hypothesized that STRAP may affect FAK activation in neuroblastoma.

Methods: The neuroblastoma cell line SK-N-AS was utilized. CRISPR-Cas9 gene editing technology was employed to establish STRAP KO cells, and STRAP OE was accomplished with stable plasmid transfection. Wild type cells (WT) and empty vector (EV) transfected cells served as controls for KO and OE cells, respectively. Immunoblotting detected protein expression. We examined the neuroblastoma patient database, GSE49710 (n=496), to investigate the abundance of STRAP and correlation with FAK in patient tumors. PamChip® kinomic peptide microarray determined the mean kinase statistics (KSTAT) and phosphorylation curves to evaluate the effects of STRAP OE on the kinome. A small molecule, PF-573,228 (PF), was used to inhibit FAK; viability and proliferation in STRAP KO and OE cells following PF treatment was examined using alamarBlue® and CellTiter96® assays, respectively.

Results: Examining the GSE49710 database, we found that higher STRAP expression was significantly associated with high-risk disease, unfavorable histology, and decreased overall survival (p ≤ 0.001). STRAP and FAK were correlated (R=0.662, p ≤ 0.001) in the patient tumors. Immunoblotting demonstrated a decrease in phosphorylated FAK (Y397) in STRAP KO cells compared to WT, indicating that STRAP may function to regulate FAK phosphorylation. Kinome studies comparing STRAP OE to EV cells found that FAK1 (KSTAT of -0.473) and FAK2 (KSTAT of -0.475) were increased. Phosphorylation activity of downstream FAK targets was increased in STRAP OE compared to EV. Following FAK inhibition with PF, STRAP OE cells had decreased viability and proliferation compared to EV as demonstrated by lower LD50s (EV 18.6 ± 3.1µM versus OE 13.0 ± 1.0µM, p=0.05) and IC50s (EV 19.4 ± 3.4µM versus OE 11.8 ± 1.4µM, p=0.03), respectively.

Conclusion: Increased STRAP expression was associated with unfavorable tumor characteristics and associated with FAK. STRAP KO in neuroblastoma led to decreased FAK phosphorylation while STRAP OE resulted in increased kinomic activity of FAK. The STRAP OE cells were more susceptible to FAK inhibition compared to the EV cells. These findings suggest a potential pathway that allows STRAP to confer a more malignant phenotype and warrants further investigation of FAK inhibition as a potential therapeutic in neuroblastomas harboring STRAP overexpression.