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CRISPR/Cas9-mediated knockout of PIM3 suppresses tumorigenesis and cancer cell stemness in human hepatoblastoma cells

Cancer Gene Therapy volume 29, pages 558–572 (2022)Cite this article

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Abstract

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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Fig. 1: PIM3 knockout decreased proliferation, viability, and motility in hepatoblastoma cells.
Fig. 2: PIM3 knockout decreased growth over time in hepatoblastoma cells and inhibited their progression through the cell cycle.
Fig. 3: RNA sequencing of PIM3 knockout and HuH6 wild-type cells.
Fig. 4: PIM3 re-introduction rescued the anti-proliferative and anti-migratory phenotype observed with PIM3 knockout.
Fig. 5: PIM3 knockout decreased hepatoblastoma cancer cell stemness.
Fig. 6: PIM3 knockout decreased in vivo tumor growth and increased animal survival in a xenograft model of hepatoblastoma.

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Acknowledgements

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.

Funding

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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  1. These authors contributed equally: Raoud Marayati, Laura L. Stafman

Authors and Affiliations

  1. Division of Pediatric Surgery, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, 35233, USA

    Raoud Marayati, Laura L. Stafman, Adele P. Williams, Laura V. Bownes, Colin H. Quinn, Hooper R. Markert, Jerry E. Stewart & Elizabeth A. Beierle

  2. Division of Transplantation, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, 35233, USA

    Juliet L. Easlick

  3. Department of Genetics, University of Alabama at Birmingham, Birmingham, AL, 35233, USA

    David K. Crossman

  4. Department of Pathology, The Children’s Hospital of Alabama, Birmingham, AL, 35233, USA

    Elizabeth Mroczek-Musulman

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Contributions

R Marayati and LL Stafman were involved in study concept and design, development of methodology, data collection, data analysis, and manuscript preparation. AP Williams, LV Bownes, CH Quinn, HR Markert, and JE Stewart contributed with data collection and analysis. JL Easlick was involved in the development of the CRISPR/Cas9 PIM3 knockout. DK Crossman performed bioinformatics analyses of the RNA sequencing data. E Mroczek-Musulman was involved in the immunohistochemistry evaluation and analysis. EA Beierle provided senior guidance with study concept and design, data analysis, and manuscript preparation.

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Correspondence to Elizabeth A. Beierle.

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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 29, 558–572 (2022). https://doi.org/10.1038/s41417-021-00334-4

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