L. L. Stafman1, A. M. Waters1, E. F. Garner1, J. E. Stewart1, E. A. Beierle1 1University Of Alabama,Birmingham, AL, USA
Introduction:
Neuroblastoma, a tumor of neural crest origin, is the most common extracranial solid tumor of childhood. For those with advanced or metastatic disease, this tumor carries a dismal prognosis. Given the current limited treatment options, novel therapies are needed. Proviral insertion site in Moloney murine leukemia virus (PIM) proteins are a family of serine/threonine kinases which has been implicated in tumorigenesis in many solid tumors including glioblastoma, but not studied extensively in neuroblastoma. We hypothesized that neuroblastoma cells would express PIM and that treatment of these cells with AZD1208 (AZD), a PIM-specific inhibitor, would lead to decreased cellular proliferation, migration, and invasion.
Methods:
Four human neuroblastoma cell lines – SK-N-AS, SK-N-BE(2), SH-EP and WAC2 – were utilized. PIM protein expression was determined by immunoblotting of whole cell lysates. PIM inhibition was accomplished with AZD, a competitive inhibitor that binds to the unique ATP pocket in PIM proteins. Proliferation was measured using CellTiter96 assays. Migration and invasion assays were completed with Transwell plates. Data reported as mean ± SEM. Student’s t-test was used to compare data between groups, with p ≤ 0.05 considered significant.
Results:
PIM protein expression was detected in all 4 neuroblastoma cell lines with immunoblotting. Inhibition of PIM with AZD (5µM, 12 hours) significantly decreased cellular proliferation in all 4 cell lines compared to control (vehicle, 12 hours) (Figure 1). In addition, PIM inhibition (AZD, 1µM) significantly inhibited cellular migration in the SK-N-AS (5872 ± 240 vs. 5000 ± 200 cells, control vs. AZD, p=0.03), SK-N-BE(2) (6580 ± 256 vs. 4182 ± 200 cells, control vs. AZD, p=0.0003), SH-EP (17388 ± 451 vs. 15960 ± 487 cells, control vs. AZD, p=0.04) and WAC2 (18200 ± 576 vs. 1660 ± 372 cells, control vs. AZD, p=0.03). Finally, AZD-induced PIM abrogation also significantly inhibited cellular invasion in the SK-N-AS (16,763 ± 559 vs. 14,868 ± 385 cells, control vs. AZD, p=0.03), SK-N-BE(2) (8872 ± 161 vs. 4909 ± 57 cells, control vs. AZD, p<0.0001) SH-EP (13,947 ± 396 vs. 6668 ± 203 cells, control vs. AZD, p<0.0001), and WAC2 (6944 ± 377 vs. 5707 ± 268 cells, control vs. AZD, p=0.04) cell lines.
Conclusion:
All four neuroblastoma cell lines utilized expressed PIM. The PIM inhibitor AZD1208 had a significant impact upon these neuroblastoma cells, resulting in decreased proliferation, migration, and invasion. These results indicate that further exploration of the PIM kinase pathway in neuroblastoma should be undertaken.
