L. Stafman1, E. Garner1, A. Hjelmeland1, J. Stewart1, S. Mruthyrunjayappa1, K. Yoon1, S. Cramer1, E. Beierle1 1University Of Alabama,Birmingham, Alabama, USA
Introduction:
Neuroblastoma (NB) is the most common extra-cranial solid tumor of childhood and is responsible for over 15% of pediatric cancer deaths. Focal adhesion kinase (FAK) is a tyrosine kinase affecting proliferation, adhesion, and migration, and is prominent in aggressive forms of NB. FAK inhibition in immortalized human NB cell lines has been shown to decrease NB tumorigenicity. Immortalized cell lines do not always recapitulate the human condition, so we have adopted the use of patient-derived xenografts (PDXs), which are tumors implanted into immunosuppressed mice from human patients. Cancer stem cells are a subpopulation of cells with stem cell-like properties. NB cancer stem cells can be identified by expression of cell surface proteins, including CD133, and the ability to grow as spheres in vitro. We hypothesized that FAK inhibition would decrease tumorigenicity and cancer stem cell maintenance in human NB PDXs.
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). FAK expression and phosphorylation was determined with immunoblotting. Viability and proliferation were assessed with alamarBlue and CellTiter 96 assays, respectively. Expression of the cell surface protein and stem cell marker, CD133, was determined using flow cytometry after 24 hours of treatment with PF or Y15. The ability of the NB PDXs to grow as spheres was assessed using an in vitro limiting dilution analysis. Student’s t-test, extreme limiting dilution analysis, and χ2 statistics were used. Data reported as mean ± SEM with p<0.05 significant.
Results:
FAK expression and phosphorylation was inhibited in both COA3 and COA6 cells following treatment with PF or Y15. FAK inhibition with the small molecules (PF or Y15) significantly decreased viability and proliferation in a dose dependent fashion in both NB PDXs. Additionally, PF or Y15 treatment yielded a significant decrease in the expression of the cancer stem cell marker, CD133 (18.5% ± 0.2 untreated cells vs. 11% ± 0.3 and 5.3% ± 0.1, PF or Y15, respectively, p< 0.02). FAK inhibition with PF or Y15 also significantly decreased sphere formation in both human NB PDXs in culture (Figure), indicating a decrease in cell “stemness”.
Conclusion:
Inhibition of FAK with small molecule inhibitors decreased viability, proliferation, and cancer stem cell maintenance in human NB PDXs. Multiple clinical trials examining the safety and efficacy of FAK inhibitors are currently ongoing, although none have yet been undertaken in children or patients with NB. Our data indicate that FAK inhibitors warrant further exploration as a novel therapy for NB.
