Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

  1. Feng J, Polychronidis G, Heger U, Frongia G, Mehrabi A, Hoffmann K. Incidence trends and survival prediction of hepatoblastoma in children: a population-based study. Cancer Commun (Lond). 2019;39:62.

    Article  Google Scholar 

  2. Zhang X, Song M, Kundu JK, Lee MH, Liu ZZ. PIM kinase as an executional target in cancer. J Cancer Prev. 2018;23:109–16.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Narlik-Grassow M, Blanco-Aparicio C, Carnero A. The PIM family of serine/threonine kinases in cancer. Med Res Rev. 2014;34:136–59.

    Article  CAS  PubMed  Google Scholar 

  4. Stafman LL, Mruthyunjayappa S, Waters AM, Garner EF, Aye JM, Stewart JE, et al. Targeting PIM kinase as a therapeutic strategy in human hepatoblastoma. Oncotarget. 2018;9:22665–79.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Popivanova BK, Li YY, Zheng H, Omura K, Fujii C, Tsuneyama K, et al. Proto-oncogene, Pim-3 with serine/threonine kinase activity, is aberrantly expressed in human colon cancer cells and can prevent Bad-mediated apoptosis. Cancer Sci. 2007;98:321–8.

    Article  CAS  PubMed  Google Scholar 

  6. Santio NM, Eerola SK, Paatero I, Yli-Kauhaluoma J, Anizon F, Moreau P, et al. Pim Kinases Promote Migration and Metastatic Growth of Prostate Cancer Xenografts. PLoS One. 2015;10:e0130340.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Morishita D, Katayama R, Sekimizu K, Tsuruo T, Fujita N. Pim kinases promote cell cycle progression by phosphorylating and down-regulating p27Kip1 at the transcriptional and posttranscriptional levels. Cancer Res. 2008;68:5076–85.

    Article  CAS  PubMed  Google Scholar 

  8. Stafman LL, Williams AP, Garner EF, Aye JM, Stewart JE, Yoon KJ, et al. Targeting PIM Kinases Affects Maintenance of CD133 Tumor Cell Population in Hepatoblastoma. Transl Oncol. 2019;12:200–8.

    Article  PubMed  Google Scholar 

  9. Stafman LL, Waldrop MG, Williams AP, Aye JM, Stewart JE, Mroczek-Musulman E, et al. The presence of PIM3 increases hepatoblastoma tumorigenesis and tumor initiating cell phenotype and is associated with decreased patient survival. J Pediatr Surg. 2019;54:1206–13.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Mocellin S, Provenzano M. RNA interference: learning gene knock-down from cell physiology. J Transl Med. 2004;2:39.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157:1262–78.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Gillory LA, Stewart JE, Megison ML, Nabers HC, Mroczek-Musulman E, Beierle EA. FAK inhibition decreases hepatoblastoma survival both in vitro and in vivo. Transl Oncol. 2013;6:206–15.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8:2281–308.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Stephenson FH, ScienceDirect. Calculations for molecular biology and biotechnology: a guide to mathematics in the laboratory, Chapter 3 - Cell Growth: Academic Press; 2010.

  15. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21.

    Article  CAS  PubMed  Google Scholar 

  16. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012;7:562–78.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010;28:511–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kramer A, Green J, Pollard J Jr, Tugendreich S. Causal analysis approaches in ingenuity pathway analysis. Bioinformatics. 2014;30:523–30.

    Article  PubMed  CAS  Google Scholar 

  19. Cheng F, Weidner-Glunde M, Varjosalo M, Rainio EM, Lehtonen A, Schulz TF, et al. KSHV reactivation from latency requires Pim-1 and Pim-3 kinases to inactivate the latency-associated nuclear antigen LANA. PLoS Pathog. 2009;5:e1000324.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Hu Y, Smyth GK. ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J Immunol Methods. 2009;347:70–8.

    Article  CAS  PubMed  Google Scholar 

  21. Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 2000;132:365–86.

    CAS  PubMed  Google Scholar 

  22. Winer J, Jung CK, Shackel I, Williams PM. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem. 1999;270:41–9.

    Article  CAS  PubMed  Google Scholar 

  23. Graham D, Magee H, Kierce B, Ball R, Dervan P, O’Meara A. Evaluation of Ki-67 reactivity in neuroblastoma using paraffin embedded tissue. Pathol Res Pr. 1995;191:87–91.

    Article  CAS  Google Scholar 

  24. Aldinucci D, Borghese C, Casagrande N. The CCL5/CCR5 axis in cancer progression. Cancers (Basel). 2020;12:1765.

    Article  CAS  Google Scholar 

  25. Baldin V, Lukas J, Marcote MJ, Pagano M, Draetta G. Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev. 1993;7:812–21.

    Article  CAS  PubMed  Google Scholar 

  26. Eichmann A, Yuan L, Breant C, Alitalo K, Koskinen PJ. Developmental expression of pim kinases suggests functions also outside of the hematopoietic system. Oncogene. 2000;19:1215–24.

    Article  CAS  PubMed  Google Scholar 

  27. Yan B, Yau EX, Samanta S, Ong CW, Yong KJ, Ng LK, et al. Clinical and therapeutic relevance of PIM1 kinase in gastric cancer. Gastric Cancer. 2012;15:188–97.

    Article  CAS  PubMed  Google Scholar 

  28. Weirauch U, Beckmann N, Thomas M, Grunweller A, Huber K, Bracher F, et al. Functional role and therapeutic potential of the pim-1 kinase in colon carcinoma. Neoplasia. 2013;15:783–94.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Bellon M, Lu L, Nicot C. Constitutive activation of Pim1 kinase is a therapeutic target for adult T-cell leukemia. Blood. 2016;127:2439–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Cohen AM, Grinblat B, Bessler H, Kristt D, Kremer A, Schwartz A, et al. Increased expression of the hPim-2 gene in human chronic lymphocytic leukemia and non-Hodgkin lymphoma. Leuk Lymphoma. 2004;45:951–5.

    Article  CAS  PubMed  Google Scholar 

  31. Li YY, Popivanova BK, Nagai Y, Ishikura H, Fujii C, Mukaida N. Pim-3, a proto-oncogene with serine/threonine kinase activity, is aberrantly expressed in human pancreatic cancer and phosphorylates bad to block bad-mediated apoptosis in human pancreatic cancer cell lines. Cancer Res. 2006;66:6741–7.

    Article  CAS  PubMed  Google Scholar 

  32. Fujii C, Nakamoto Y, Lu P, Tsuneyama K, Popivanova BK, Kaneko S, et al. Aberrant expression of serine/threonine kinase Pim-3 in hepatocellular carcinoma development and its role in the proliferation of human hepatoma cell lines. Int J Cancer. 2005;114:209–18.

    Article  CAS  PubMed  Google Scholar 

  33. Li YY, Mukaida N. Pathophysiological roles of Pim-3 kinase in pancreatic cancer development and progression. World J Gastroenterol. 2014;20:9392–404.

    PubMed  PubMed Central  Google Scholar 

  34. Mikkers H, Nawijn M, Allen J, Brouwers C, Verhoeven E, Jonkers J, et al. Mice deficient for all PIM kinases display reduced body size and impaired responses to hematopoietic growth factors. Mol Cell Biol. 2004;24:6104–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. van der Lugt NM, Domen J, Verhoeven E, Linders K, van der Gulden H, Allen J, et al. Proviral tagging in E mu-myc transgenic mice lacking the Pim-1 proto-oncogene leads to compensatory activation of Pim-2. EMBO J. 1995;14:2536–44.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Qu Y, Zhang C, Du E, Wang A, Yang Y, Guo J, et al. Pim-3 is a critical risk factor in development and prognosis of prostate cancer. Med Sci Monit. 2016;22:4254–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Zhuang H, Zhao MY, Hei KW, Yang BC, Sun L, Du X, et al. Aberrant expression of pim-3 promotes proliferation and migration of ovarian cancer cells. Asian Pac J Cancer Prev. 2015;16:3325–31.

    Article  PubMed  Google Scholar 

  38. Liu B, Wang Z, Li HY, Zhang B, Ping B, Li YY. Pim-3 promotes human pancreatic cancer growth by regulating tumor vasculogenesis. Oncol Rep. 2014;31:2625–34.

    Article  PubMed  CAS  Google Scholar 

  39. Leroux AE, Biondi RM. Renaissance of allostery to disrupt protein kinase interactions. Trends Biochem Sci. 2020;45:27–41.

    Article  CAS  PubMed  Google Scholar 

  40. Nair JR, Caserta J, Belko K, Howell T, Fetterly G, Baldino C, et al. Novel inhibition of PIM2 kinase has significant anti-tumor efficacy in multiple myeloma. Leukemia. 2017;31:1715–26.

    Article  CAS  PubMed  Google Scholar 

  41. Narlik-Grassow M, Blanco-Aparicio C, Cecilia Y, Peregrina S, Garcia-Serelde B, Munoz-Galvan S, et al. The essential role of PIM kinases in sarcoma growth and bone invasion. Carcinogenesis. 2012;33:1479–86.

    Article  CAS  PubMed  Google Scholar 

  42. Garcia PD, Langowski JL, Wang Y, Chen M, Castillo J, Fanton C, et al. Pan-PIM kinase inhibition provides a novel therapy for treating hematologic cancers. Clin Cancer Res. 2014;20:1834–45.

    Article  CAS  PubMed  Google Scholar 

  43. Sharpe JJ, Cooper TA. Unexpected consequences: exon skipping caused by CRISPR-generated mutations. Genome Biol. 2017;18:109.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Joberty G, Falth-Savitski M, Paulmann M, Bosche M, Doce C, Cheng AT, et al. A tandem guide RNA-based strategy for efficient CRISPR gene editing of cell populations with low heterogeneity of edited alleles. CRISPR J. 2020;3:123–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Zhang XH, Tee LY, Wang XG, Huang QS, Yang SH. Off-target effects in CRISPR/Cas9-mediated genome engineering. Mol Ther Nucleic Acids. 2015;4:e264.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Ma H, Dang Y, Wu Y, Jia G, Anaya E, Zhang J, et al. A CRISPR-based screen identifies genes essential for west-nile-virus-induced cell death. Cell Rep. 2015;12:673–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Heck JE, Meyers TJ, Lombardi C, Park AS, Cockburn M, Reynolds P, et al. Case-control study of birth characteristics and the risk of hepatoblastoma. Cancer Epidemiol. 2013;37:390–5.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Turusov VS, Torii M, Sills RC, Willson GA, Herbert RA, Hailey JR, et al. Hepatoblastomas in mice in the US National Toxicology Program (NTP) studies. Toxicol Pathol. 2002;30:580–91.

    Article  CAS  PubMed  Google Scholar 

  49. Horton JD, Lee S, Brown SR, Bader J, Meier DE. Survival trends in children with hepatoblastoma. Pediatr Surg Int. 2009;25:407–12.

    Article  PubMed  Google Scholar 

  50. Mertens B, Cristina de Araujo Nogueira T, Topalis D, Stranska R, Snoeck R, Andrei G. Investigation of tumor-tumor interactions in a double human cervical carcinoma xenograft model in nude mice. Oncotarget. 2018;9:21978–2000.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Wu Y, Wang YY, Nakamoto Y, Li YY, Baba T, Kaneko S, et al. Accelerated hepatocellular carcinoma development in mice expressing the Pim-3 transgene selectively in the liver. Oncogene. 2010;29:2228–37.

    Article  CAS  PubMed  Google Scholar 

  52. Chang JC. Cancer stem cells: role in tumor growth, recurrence, metastasis, and treatment resistance. Medicine (Baltim). 2016;95:S20–5.

    Article  CAS  Google Scholar 

  53. Jensen JB, Parmar M. Strengths and limitations of the neurosphere culture system. Mol Neurobiol. 2006;34:153–61.

    Article  CAS  PubMed  Google Scholar 

  54. Villodre ES, Kipper FC, Pereira MB, Lenz G. Roles of OCT4 in tumorigenesis, cancer therapy resistance and prognosis. Cancer Treat Rev. 2016;51:1–9.

    Article  CAS  PubMed  Google Scholar 

  55. Iv Santaliz-Ruiz LE, Xie X, Old M, Teknos TN, Pan Q. Emerging role of nanog in tumorigenesis and cancer stem cells. Int J Cancer. 2014;135:2741–8.

    Article  CAS  PubMed  Google Scholar 

  56. Mamun MA, Mannoor K, Cao J, Qadri F, Song X. SOX2 in cancer stemness: tumor malignancy and therapeutic potentials. J Mol Cell Biol. 2020;12:85–98.

    Article  PubMed  CAS  Google Scholar 

  57. Neradil J, Veselska R. Nestin as a marker of cancer stem cells. Cancer Sci. 2015;106:803–11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Bahnassy AA, Fawzy M, El-Wakil M, Zekri AR, Abdel-Sayed A, Sheta M. Aberrant expression of cancer stem cell markers (CD44, CD90, and CD133) contributes to disease progression and reduced survival in hepatoblastoma patients: 4-year survival data. Transl Res. 2015;165:396–406.

    Article  CAS  PubMed  Google Scholar 

  59. Rikhi RR, Spady KK, Hoffman RI, Bateman MS, Bateman M, Howard LE. Hepatoblastoma: a need for cell lines and tissue banks to develop targeted drug therapies. Front Pediatr. 2016;4:22.

    Article  PubMed  PubMed Central  Google Scholar 

  60. Costantini S, Di Bernardo G, Cammarota M, Castello G, Colonna G. Gene expression signature of human HepG2 cell line. Gene. 2013;518:335–45.

    Article  CAS  PubMed  Google Scholar 

Download references

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).

Author information

Author notes

  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

Authors
  1. Raoud Marayati
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Laura L. Stafman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Adele P. Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Laura V. Bownes
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Colin H. Quinn
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hooper R. Markert
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Juliet L. Easlick
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jerry E. Stewart
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. David K. Crossman
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Elizabeth Mroczek-Musulman
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Elizabeth A. Beierle
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding author

Correspondence to Elizabeth A. Beierle.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41417-021-00334-4

This article is cited by

Search

Advanced search

Quick links