J. Aye1, S. Mruthyunjayappa1, L. Stafman1, E. Garner1, J. Stewart1, E. Mroczek-Musulman1, K. Yoon1, K. Whelan1, E. Beierle1 1University Of Alabama at Birmingham,Birmingham, Alabama, USA
Introduction:
Approximately 12% of patients with Wilms tumor (WT) will have metastatic disease at diagnosis and often have a grave prognosis. We established a patient-derived xenograft (PDX) of metastatic WT, including a liver metastasis (COA 42), along with its matched isogenic primary renal tumor (COA 25). Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase involved in the tumorigenesis of pediatric renal tumors. To date, the role of FAK in metastatic WT has not been investigated. We hypothesized FAK inhibition would decrease tumorigenicity in this PDX model.
Methods:
Two human WT samples, primary renal tumor, COA 25, and hepatic metastasis, COA 42, were implanted in athymic nude mice. H&E staining of the patient’s renal primary and liver metastasis and subsequent PDXs, COA 25 and COA 42, was performed. FAK expression and phosphorylation was detected with immunohistochemical staining (IHC) of patient samples and by immunoblotting of PDX protein lysate. COA 25 and COA 42 cells were treated with small molecule FAK inhibitors, PF-573228 (PF) and 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15), for 24 hours. Cell viability and proliferation were assessed with alamarBlue and CellTiter 96 assays, respectively. Cell cycle analysis was performed by flow cytometry. Results were compared with student’s t-test and p≤0.05 was considered significant.
Results:
H&E staining confirmed COA 25 and COA 42 recapitulated the patient’s primary and metastatic tumor, respectively, and IHC confirmed FAK expression and phosphorylation in these tissues. Immunoblotting revealed FAK was present and phosphorylated in both PDXs. FAK inhibition with PF (10 μM) significantly decreased cell viability by 63% and proliferation by 47% in the COA 25 cells (renal primary) compared to untreated cells. Y15-induced FAK inhibition (10 μM) of COA 25 cells also significantly decreased cell survival by 61% and proliferation by 36%. Treatment of the COA 42 cells (hepatic metastasis) with PF and Y15 (10 μM) also significantly reduced cell survival by 83% and 68%, respectively, and proliferation by 63% and 72%, respectively. Flow cytometry revealed G1 cell cycle arrest for both COA 25 and COA 42 treated with PF and Y15 (10 μM) compared to untreated cells (Figure).
Conclusion:
FAK protein was expressed and phosphorylated in primary renal and metastatic WT PDXs. FAK inhibition with two small molecules led to decreased tumor cell viability and proliferation along with cell cycle arrest in both the primary renal tumor and liver metastasis. These findings suggest that further exploration of FAK as a therapeutic target for metastatic WT should be undertaken.
