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Sleeping Beauty transposon mutagenesis in mouse intestinal organoids identifies genes involved in tumor progression and metastasis

Cancer Gene Therapy volume 31, pages 527–536 (2024)Cite this article

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Abstract

To identify genes important for colorectal cancer (CRC) development and metastasis, we established a new metastatic mouse organoid model using Sleeping Beauty (SB) transposon mutagenesis. Intestinal organoids derived from mice carrying actively mobilizing SB transposons, an activating KrasG12D, and an inactivating ApcΔ716 allele, were transplanted to immunodeficient mice. While 66.7% of mice developed primary tumors, 7.6% also developed metastatic tumors. Analysis of SB insertion sites in tumors identified numerous candidate cancer genes (CCGs) identified previously in intestinal SB screens performed in vivo, in addition to new CCGs, such as Slit2 and Atxn1. Metastatic tumors from the same mouse were clonally related to each other and to primary tumors, as evidenced by the transposon insertion site. To provide functional validation, we knocked out Slit2, Atxn1, and Cdkn2a in mouse tumor organoids and transplanted to mice. Tumor development was promoted when these gene were knocked out, demonstrating that these are potent tumor suppressors. Cdkn2a knockout cells also metastasized to the liver in 100% of the mice, demonstrating that Cdkn2a loss confers metastatic ability. Our organoid model thus provides a new approach that can be used to understand the evolutionary forces driving CRC metastasis and a rich resource to uncover CCGs promoting CRC.

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Fig. 1: Establishment of a mouse model for colorectal cancer metastasis.
Fig. 2: Histological analyses of SB-induced tumors.
Fig. 3: Analyses of CISs and validation of CCGs.
Fig. 4: Validation of candidate tumor suppressor genes.
Fig. 5: Validation of candidate metastasis suppressor genes.

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Data availability

The RNA-seq data in this study have been deposited in the Gene Expression Omnibus (GEO) database under accession codes GSE248668.

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Acknowledgements

We thank the TCGA Research Network for sharing the CRC somatic mutational data. We also thank S. Kataoka, A. Tsuda, M. Watanabe, Y. Jomen and K. Sato for technical assistance. Histological sections were made by Y. Shiotani in the Core facility of National Cancer Center Research Institute. This study was supported by FOREST (JPMJFR2164), JSPS (21K19421, 20H03522), the National Cancer Center Research and Development Fund (2020-A-5), Princess Takamatsu Cancer Fund, Takeda Science Foundation, AMED (23ama221529h0001) and JH (2022-B-02).

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Author notes

  1. These authors contributed equally: Naoko Iida, Yukari Muranaka

Authors and Affiliations

  1. Division of Genome Analysis Platform Development, National Cancer Center Research Institute, Tokyo, Japan

    Naoko Iida & Yuichi Shiraishi

  2. Laboratory of Molecular Genetics, National Cancer Center Research Institute, Tokyo, Japan

    Yukari Muranaka & Haruna Takeda

  3. Division of Biomedical Convergence, College of Biomedical Science, Kang-won National University, Chuncheon-si, Republic of Korea

    Jun Won Park

  4. Division of Molecular Pathology, National Cancer Center Research Institute, Tokyo, Japan

    Shigeki Sekine

  5. Genetics Department, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Neal G. Copeland & Nancy A. Jenkins

  6. Division of Genetics, Cancer Research Institute, Kanazawa University, Ishikawa, Japan

    Masanobu Oshima

  7. Nano-Life Science Institute, Kanazawa University, Ishikawa, Japan

    Masanobu Oshima

  8. Cancer genes and genomes unit, Cancer Research Institute, Kanazawa University, Ishikawa, Japan

    Haruna Takeda

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Contributions

NI and YS established the informatics pipeline for SB screening and generated the SB data. YM and J-WP performed the animal experiments, analyzed the data. MO provided the mouse lines and established organoids. SS diagnosed the tumor. NAJ and NGC provided the mouse lines and wrote the manuscript. HT designed the experiment, analyzed the data, and wrote the manuscript.

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Correspondence to Haruna Takeda.

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Iida, N., Muranaka, Y., Park, J.W. et al. Sleeping Beauty transposon mutagenesis in mouse intestinal organoids identifies genes involved in tumor progression and metastasis. Cancer Gene Ther 31, 527–536 (2024). https://doi.org/10.1038/s41417-023-00723-x

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