E. F. Garner1,2, A. M. Waters1, L. L. Stafman1, J. E. Stewart1, E. Mroczek-Musulman2, E. A. Beierle1 1University Of Alabama,Pediatric Surgery,Birmingham, Alabama, USA 2University Of Alabama,Pathology,Birmingham, Alabama, USA
Introduction:
Neuroblastoma is the most common extra-cranial solid tumor in children. Despite advances in therapy, the outcomes for children with advanced-staged disease remain dismal. The role of protein phosphatase 2A (PP2A) as a tumor suppressor has been characterized in other cancers. We hypothesized that increasing tumor suppressor PP2A activity would inhibit tumorigenesis in neuroblastoma cells.
Methods:
Institution approval was obtained from IRB (X111123007) and IACUC (09064). Immunohistochemical staining for the endogenous PP2A inhibitor, I2PP2A, was completed on 25 human neuroblastoma specimens. Slides were scored by a blinded pathologist and a stain score calculated based upon the formula of percent of cells staining positive (0-100%) × intensity of the stain (0-3) resulting in a score from 0-300. Human neuroblastoma cell lines SH-EP and WAC2 were utilized. Activation of PP2A was accomplished with multiple approaches: transient inhibition of the endogenous inhibitor (I2PP2A) with siRNA and stable knockdown with shRNA; non-specific activation with forskolin; direct activation with small molecule FTY720. Migration and invasion assays were performed with Transwell plates. Proliferation was measured using CellTiter96 assay. Stable I2PP2A knockdown clones in the SH-EP and WAC2 cell lines were used for in vivo studies. Empty vector (shEV) control and shI2PP2A cells (2.5 × 106 cells) were injected into the right flank of nude mice (N=5/group) and animals monitored for tumor growth for 2 months. Data reported as median (range) or mean ± SEM and Student’s t-test was used to compare data between groups, with p ≤ 0.05 considered significant.
Results:
I2PP2A staining was present in 22 of the 25 human specimens examined and the median stain score was 140 (0-180), indicating presence of the endogenous PP2A inhibitor. Protein for I2PP2A and PP2A was detected in both neuroblastoma cell lines. Transient inhibition of I2PP2A with siRNA significantly decreased migration and invasion in both cell lines. Further, small molecule activation of PP2A with forskolin or FTY720 significantly decreased proliferation in both cell lines. Finally, stable clones with knock-down of I2PP2A, the endogenous PP2A inhibitor, allowing activation of PP2A, showed significantly decreased tumor growth in both the SH-EP (1211 ± 587 mm3 vs. 347 ± 149 mm3, shEV vs. shI2PP2A, p=0.04) and WAC2 (1714 ± 210 mm3 vs. 1031 ± 139 mm3, shEV vs. shI2PP2A, p=0.01) cell lines.
Conclusion:
The activation of PP2A in neuroblastoma cell lines by inhibition of the endogenous inhibitor (I2PP2A) and direct activation with forskolin or FTY720 significantly inhibited cellular proliferation, migration and invasion. Also, knockdown of the PP2A inhibitor, I2PP2A, led to inhibition of neuroblastoma tumor growth in vivo. These results suggest a role for activation of PP2A tumor suppressor as a novel therapeutic strategy for neuroblastoma.