H. R. Markert1, R. Marayati1, A. P. Williams1, C. H. Quinn1, J. E. Stewart1, E. A. Beierle1 1University Of Alabama at Birmingham,Division Of Pediatric Surgery, Department Of Surgery,Birmingham, Alabama, USA
Introduction: Hepatoblastoma is the most common liver tumor in children. Those with relapsed or metastatic disease continue to have a poor prognosis and will require novel therapies to improve outcomes. We have previously identified that Proviral Integration site in Maloney murine leukemia virus (PIM3) kinase plays a role in hepatoblastoma tumorigenesis. PIM447 has shown promise as a targeted PIM3 kinase inhibitor. We hypothesized that treatment of hepatoblastoma cells with PIM447 would result in decreased tumorigenesis in vitro.
Methods: The human hepatoblastoma cell line HuH6 was examined in monolayer culture conditions. Cells were treated with PIM447 at increasing concentrations for 72 hours. Cell viability and proliferation were determined using alamarBlue® and CellTiter96® assays, respectively. Migration and invasion were evaluated using 8µm micropore transwell inserts coated with collagen and MatrigelTM, respectively. Migration was also assessed using a cell monolayer wounding assay. Western blotting was used to measure protein expression of PIM kinases and their known downstream targets, BAD and phosphorylated BAD (pBAD), as well as apoptotic markers cleaved PARP and cleaved caspase 3. Experiments were repeated in triplicate, and statistical analysis was performed with Student’s t-test and reported as a mean ± SEM, with p ≤ 0.05 deemed significant.
Results: PIM inhibition with PIM447 led to decreased cell viability and proliferation compared to untreated HuH6 cells (figure 1A, B). Both migration and invasion were decreased after treatment with PIM447 (figure 1C, D). Cell monolayer wounding assay demonstrated a significant decrease in cell motility with PIM447 treatment (figure 1E, F). Immunoblotting confirmed the presence and subsequent downregulation of downstream PIM targets BAD and pBAD and upregulation of apoptotic markers cleaved PARP and cleaved caspase 3 after treatment with PIM447.
Conclusions: PIM447 reduced viability, proliferation, and motility in HuH6 human hepatoblastoma cells and suppressed downstream targets of PIM3. These data suggest PIM447 should be further investigated as a potential novel therapeutic in human hepatoblastoma.
