Hepatoblastoma remains one of the most difficult childhood tumors to treat and is alarmingly understudied. We previously demonstrated that Proviral Insertion site in Maloney murine leukemia virus (PIM) kinases, specifically PIM3, are overexpressed in human hepatoblastoma cells and function to promote tumorigenesis. We aimed to use CRISPR/Cas9 gene editing with dual gRNAs to introduce large inactivating deletions in the PIM3 gene and achieve stable PIM3 knockout in the human hepatoblastoma cell line, HuH6. PIM3 knockout of hepatoblastoma cells led to significantly decreased proliferation, viability, and motility, inhibited cell-cycle progression, decreased tumor growth in a xenograft murine model, and increased animal survival. Analysis of RNA sequencing data revealed that PIM3 knockout downregulated expression of pro-migratory and pro-invasive genes and upregulated expression of genes involved in apoptosis and differentiation. Furthermore, PIM3 knockout decreased hepatoblastoma cancer cell stemness as evidenced by decreased tumorsphere formation, decreased mRNA abundance of stemness markers, and decreased cell surface expression of CD133, a marker of hepatoblastoma stem cell-like cancer cells. Reintroduction of PIM3 into PIM3 knockout cells rescued the malignant phenotype. Successful CRISPR/Cas9 knockout of PIM3 kinase in human hepatoblastoma cells confirmed the role of PIM3 in promoting hepatoblastoma tumorigenesis and cancer cell stemness.
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We wish to thank Vidya Sagar Hanumanthu and the UAB Comprehensive Flow Cytometry Core (supported by NIH P30 AR048311 and NIH P30 AI27667), Dr. Michael Crowley and the UAB Genomics Core (supported by NIH CA013148), and Dr. Anita Hjemeland’s laboratory for their assistance with the qPCR.
This project was made possible by funding from the National Cancer Institute of the National Institutes of Health under award numbers T32 CA229102 (RM and LVB), T32 CA091078 (LLS), T32 CA183926 (APW), 5T32GM008361 (CHQ), P30 AR048311, and P30 AI027767 to the Flow Cytometry Core, and CA013148 to the UAB Genomics Core. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Other funding sources include Cannonball Kids cancer, Sid Strong Foundation, Elaine Roberts Foundation, Open Hands Overflowing Hearts, and Starr Fund-Vince Lombardi Cancer Foundation (EAB), and the Society of University Surgeons (RM).
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The authors declare no competing interests.
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Marayati, R., Stafman, L.L., Williams, A.P. et al. CRISPR/Cas9-mediated knockout of PIM3 suppresses tumorigenesis and cancer cell stemness in human hepatoblastoma cells. Cancer Gene Ther (2021). https://doi.org/10.1038/s41417-021-00334-4