L. Stafman1, A. P. Williams1, J. Aye1, J. Stewart1, E. A. Beierle1 1University Of Alabama at Birmingham,Birmingham, Alabama, USA
Introduction:
Despite increasing incidence, treatment for hepatoblastoma has not changed significantly over the past 20 years. Treatment strategies primarily rely on cisplatin, but chemoresistance remains a significant challenge. Stem cell-like cancer cells (SCLCCs) are a subset of cancer cells thought to play a role in chemoresistance. We have previously demonstrated that Proviral Integration site for Moloney murine leukemia (PIM) kinases, specifically PIM3, play a role in hepatoblastoma and decrease the SCLCC phenotype, and that the combination of PIM inhibition with cisplatin acts synergistically to decrease hepatoblastoma cell proliferation. We therefore sought to develop a cisplatin resistance model and evaluate the effect of cisplatin and PIM inhibition in combination on the SCLCC phenotype and the effect of PIM inhibition on cisplatin-resistant hepatoblastoma cells.
Methods:
Cisplatin-resistant human hepatoblastoma cells were developed through serial passage of the human hepatoblastoma HuH6 cells in athymic nude mice and treatment of the mice with cisplatin (2 mg/kg/day three consecutive days weekly). PIM inhibition was achieved using the small molecule pan-PIM inhibitor, AZD1208. Cell viability and proliferation were assessed using the alamarBlue® and CellTiter 96® colorimetric assays. Combination indices were calculated and isobolograms constructed using the method of Chou and Talalay. CD133 expression was determined using flow cytometry and an extreme limiting dilution analysis was used to determine sphere frequency. PIM3 expression was assessed by Western blotting.
Results:
Cell viability and proliferation in the presence of cisplatin was increased in the cisplatin-resistant cells compared to cisplatin-naïve cells, confirming the cisplatin resistance model. Cisplatin-resistant hepatoblastoma cells both expressed more CD133 and formed tumorspheres more readily than cisplatin-naïve cells, indicating an increase in the SCLCC phenotype. Cisplatin-resistant hepatoblastoma cells exhibited higher PIM3 expression. Finally, PIM inhibition in combination with cisplatin resulted in combination indices of 0.25 and 0.35, indicating that PIM inhibition sensitized even cisplatin-resistant cells to cisplatin (Figure).
Conclusion:
Together, these findings provide evidence that PIM inhibition may be promising in combination with the standard chemotherapeutic cisplatin in hepatoblastoma to target SCLCCs and prevent chemoresistance to cisplatin.
