L. Stafman1, E. Garner1, J. Aye1, A. Williams1, J. Stewart1, K. Yoon1, K. Whelan1, E. Beierle1 1University Of Alabama at Birmingham,Birmingham, Alabama, USA
Introduction:
Neuroblastoma (NB) is the most common extracranial solid tumor of childhood and despite aggressive therapies, over 50% of patients with high risk tumors will suffer disease relapse. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase present in aggressive NB. Tumor initiating cells (TICs) are cancer cells with stem cell-like properties thought to be responsible for tumor recurrence and chemoresistance. We previously demonstrated that FAK inhibition decreased CD133 expression and tumorsphere formation, both markers of TICs, in NB patient-derived xenografts (PDXs). We hypothesized that targeting FAK would decrease stemness and tumorigenicity in NB TIC population.
Methods:
Cells from two human NB PDXs (COA3, COA6) were treated with the FAK inhibitors 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15) or PF-573,228 (PF). Expression of TIC markers, Sox2, Oct4, and Nanog, were assessed by immunoblotting. Cells were separated by CD133 surface expression using microbeads to obtain TIC (CD133-enriched) and non-TIC (CD133-depleted) populations. Tumorsphere formation assays were performed. Invasion was evaluated using Transwell inserts. Cell cycle analyses were performed by flow cytometry. Student’s t-test, extreme limiting dilution analysis, and χ 2 statistics were used with mean ± standard error of the mean reported and p ≤ 0.05 significant.
Results:
FAK inhibition with PF or Y15 significantly decreased expression of the TIC markers Sox, Oct4, and Nanog in NB PDXs. Additionally, treatment with PF or Y15 decreased tumorsphere formation in both TICs and non-TICs (p<0.01). Invasion was significantly decreased with FAK inhibition in both TIC (98% ± 2% decrease with 5 μ M PF, 86% ± 11% decrease with 5 μM Y15) and non-TIC (96% ± 1% decrease with 5 μM PF, 62% ± 9% decrease with 5 μM Y15) populations. FAK inhibition with both small molecules resulted in significantly decreased invasion in both the TIC and non-TIC cell populations. FAK inhibition also led to cell cycle arrest with significant increases in G1 and decreases in S phase of the cell cycle in both TICs and non-TICS (Figure).
Conclusion:
Inhibition of FAK with small molecule inhibitors decreased expression of TIC markers in human NB PDXs. NB TICs (CD133-enriched cells) were not immune to FAK inhibition and exhibited decreased tumorsphere formation, invasion, and cell cycle arrest similar to their non-TIC (CD133-depleted) counterparts. These findings suggest that FAK supports the NB TIC phenotype and targeting FAK in this cell population may be an important therapeutic strategy to decrease NB chemoresistance and relapse.
