R. Marayati1, L. L. Stafman1, A. P. Williams1, J. L. Easlick2, H. R. Markert1, J. M. Aye3, J. E. Stewart1, E. A. Beierle1 1University Of Alabama at Birmingham,Division Of Pediatric Surgery, Department Of Surgery,Birmingham, Alabama, USA 2University Of Alabama at Birmingham,Division Of Transplantation, Department Of Surgery,Birmingham, Alabama, USA 3University Of Alabama at Birmingham,Division Of Pediatric Hematology Oncology, Department Of Pediatrics,Birmingham, Alabama, USA
Introduction: Hepatoblastoma (HB) is the most common primary liver tumor in children. Despite an increasing incidence and major advances in care for other pediatric solid tumors, therapy for HB has remained virtually unchanged for the last 20 years. Over half of patients initially present with metastatic or advanced disease and the prognosis for these children remains dismal. We previously demonstrated that Proviral Insertion site in Maloney murine leukemia virus (PIM) kinases, specifically PIM3, are overexpressed in human HB cells and function to promote tumorigenesis. CRISPR-Cas9 gene editing technology is a powerful approach for loss of function studies. We aimed to use CRISPR-Cas9 mediated PIM3 knockout (KO) to confirm and verify the role of PIM3 kinase in promoting cell proliferation and survival and further characterize the effects of PIM3 KO on migration and invasion of HB in vitro as distinct early steps of the metastatic cascade.
Methods: CRISPR-Cas9 gene editing technology was used to introduce inactivating deletions in the PIM3 gene and achieve stable PIM3 KO in the long-term passaged human HB cell line, HuH6. PIM3 gene editing was confirmed by PCR and Sanger sequencing and resulting knockout of protein expression by immunoblotting. Cell proliferation and viability were measured using the CellTiter 96® and alamarBlue® colorimetric assays, respectively. Migration and invasion were evaluated using modified Boyden chambers with 8 µm micropore Transwell inserts coated with collagen and MatrigelTM (BD Biosciences) respectively. Student’s t-test was used with mean ± standard error of the mean reported and p≤0.05 significant.
Results: Immunoblotting confirmed expression and stable KO of PIM3 in the HuH6 human HB cell line. PIM3 KO HuH6 cells exhibited a significant decrease in proliferation (18.4%, p=0.024) and viability (27.1%, p<0.001) compared to control HuH6 cells. Migration was significantly decreased with PIM3 KO (983 ± 132 vs 375 ± 46 cells, control vs PIM3 KO, p<0.001). Additionally, PIM3 KO resulted in significantly decreased invasion through Matrigel (153 ± 46 vs 63 ± 30 cells, control vs PIM3 KO, p=0.002).
Conclusion: Stable CRISPR-Cas9 PIM3 KO leads to decreased cell proliferation, viability, migration, and invasion of human HB cells. These findings suggest that PIM3 promotes the metastatic phenotype in HB in vitro and that targeting PIM3 may provide a novel therapeutic strategy for children with metastatic or relapsed disease.
