• Open Access

Identification of a lung adenocarcinoma cell line with CCDC6-RET fusion gene and the effect of RET inhibitors in vitro and in vivo

Authors

  • Makito Suzuki,

    1. Division of Translational Research, Research Center for Innovative Oncology, National Cancer Center Hospital East, Kashiwa, Chiba, Japan
    2. Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan
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    • These authors contributed equally to this work.
  • Hideki Makinoshima,

    1. Division of Translational Research, Research Center for Innovative Oncology, National Cancer Center Hospital East, Kashiwa, Chiba, Japan
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    • These authors contributed equally to this work.
  • Shingo Matsumoto,

    1. Division of Translational Research, Research Center for Innovative Oncology, National Cancer Center Hospital East, Kashiwa, Chiba, Japan
    2. Division of Thoracic Oncology, National Cancer Center Hospital East, Kashiwa, Chiba, Japan
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  • Ayako Suzuki,

    1. Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan
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  • Sachiyo Mimaki,

    1. Division of Translational Research, Research Center for Innovative Oncology, National Cancer Center Hospital East, Kashiwa, Chiba, Japan
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  • Koutatsu Matsushima,

    1. Division of Translational Research, Research Center for Innovative Oncology, National Cancer Center Hospital East, Kashiwa, Chiba, Japan
    2. Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan
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  • Kiyotaka Yoh,

    1. Division of Thoracic Oncology, National Cancer Center Hospital East, Kashiwa, Chiba, Japan
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  • Koichi Goto,

    1. Division of Thoracic Oncology, National Cancer Center Hospital East, Kashiwa, Chiba, Japan
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  • Yutaka Suzuki,

    1. Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan
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  • Genichiro Ishii,

    1. Division of Pathology, Research Center for Innovative Oncology, National Cancer Center Hospital East, Kashiwa, Chiba, Japan
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  • Atsushi Ochiai,

    1. Division of Pathology, Research Center for Innovative Oncology, National Cancer Center Hospital East, Kashiwa, Chiba, Japan
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  • Koji Tsuta,

    1. Division of Pathology and Clinical Laboratories, National Cancer Center Hospital, Tokyo, Japan
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  • Tatsuhiro Shibata,

    1. Division of Cancer Genomics, National Cancer Center Research Institute, Tokyo, Japan
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  • Takashi Kohno,

    1. Division of Genome Biology, National Cancer Center Research Institute, Tokyo, Japan
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  • Hiroyasu Esumi,

    1. National Cancer Center Hospital East, Kashiwa, Chiba, Japan
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  • Katsuya Tsuchihara

    Corresponding author
    1. Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan
    • Division of Translational Research, Research Center for Innovative Oncology, National Cancer Center Hospital East, Kashiwa, Chiba, Japan
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To whom correspondence should be addressed.

E-mail: ktsuchih@east.ncc.go.jp

Abstract

Rearrangements of the proto-oncogene RET are newly identified potential driver mutations in lung adenocarcinoma (LAD). However, the absence of cell lines harboring RET fusion genes has hampered the investigation of the biological relevance of RET and the development of RET-targeted therapy. Thus, we aimed to identify a RET fusion positive LAD cell line. Eleven LAD cell lines were screened for RET fusion transcripts by reverse transcription-polymerase chain reaction. The biological relevance of the CCDC6-RET gene products was assessed by cell growth, survival and phosphorylation of ERK1/2 and AKT with or without the suppression of RET expression using RNA interference. The efficacy of RET inhibitors was evaluated in vitro using a culture system and in an in vivo xenograft model. Expression of the CCDC6-RET fusion gene in LC-2/ad cells was demonstrated by the mRNA and protein levels, and the genomic break-point was confirmed by genomic DNA sequencing. Mutations in KRAS and EGFR were not observed in the LC-2/ad cells. CCDC6-RET was constitutively active, and the introduction of a siRNA targeting the RET 3' region decreased cell proliferation by downregulating RET and ERK1/2 phosphorylation. Moreover, treatment with RET-inhibitors, including vandetanib, reduced cell viability, which was accompanied by the downregulation of the AKT and ERK1/2 signaling pathways. Vandetanib exhibited anti-tumor effects in the xenograft model. Endogenously expressing CCDC6-RET contributed to cell growth. The inhibition of kinase activity could be an effective treatment strategy for LAD. LC-2/ad is a useful model for developing fusion RET-targeted therapy.

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