Cytoplasmic accumulation of connexin32 expands cancer stem cell population in human HuH7 hepatoma cells by enhancing its self-renewal

Authors

  • Yohei Kawasaki,

    1. Department of Molecular and Tumour Pathology, Akita University Graduate School of Medicine, Akita, Japan
    2. Department of Otorhinolaryngology, Akita University Graduate School of Medicine, Akita, Japan
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  • Yasufumi Omori,

    Corresponding author
    1. Department of Molecular and Tumour Pathology, Akita University Graduate School of Medicine, Akita, Japan
    • Department of Molecular and Tumour Pathology, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan
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    • Tel: +81-18-884-6061, Fax: +81-18-836-2601

  • Qingchang Li,

    1. Department of Molecular and Tumour Pathology, Akita University Graduate School of Medicine, Akita, Japan
    2. Department of Pathology, College of Basic Medical Sciences, China Medical University, Shenyang, China
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  • Yuji Nishikawa,

    1. Department of Molecular and Tumour Pathology, Akita University Graduate School of Medicine, Akita, Japan
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  • Toshiaki Yoshioka,

    1. Department of Molecular and Tumour Pathology, Akita University Graduate School of Medicine, Akita, Japan
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  • Masayuki Yoshida,

    1. Department of Molecular and Tumour Pathology, Akita University Graduate School of Medicine, Akita, Japan
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  • Kazuo Ishikawa,

    1. Department of Otorhinolaryngology, Akita University Graduate School of Medicine, Akita, Japan
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  • Katsuhiko Enomoto

    1. Department of Molecular and Tumour Pathology, Akita University Graduate School of Medicine, Akita, Japan
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Abstract

Although the connexin32 (Cx32)-mediated gap junction is abolished in hepatocellular carcinoma (HCC), the expression of cytoplasmic Cx32 tends to increase in correspondence with the grade of malignancy. Establishing a Tet-off expression system in human nonmetastatic HuH7 HCC cells where cytoplasmic Cx32 was overexpressed by doxycycline (Dox) withdrawal, we previously demonstrated that overexpression of cytoplasmic Cx32 made HuH7 cells metastatic in mice. In our study, hypothesizing that the cytoplasmic Cx32-induced metastasis may involve expansion of the cancer stem cell (CSC) population, we examined whether cytoplasmic Cx32 controlled the size of the side population (SP) in HuH7 Tet-off Cx32 cells. Fluorescence-activated cell sorting revealed that SP was expanded in a Dox-free medium compared with a Dox-supplemented one. Although cytoplasmic Cx32 did not block maturation from SP to non-SP, purified SP reconstituted a larger SP fraction in the Dox-free medium than in the Dox-supplemented one. Furthermore, although SP from HuH7 Tet-off mock cells formed a similar number of CSC spheres of a similar size whether with or without Dox, SP from HuH7 Tet-off Cx32 cells developed a greater number of larger CSC spheres in the Dox-free medium than in the Dox-supplemented one. Taken together, these results suggest that accumulation of cytoplasmic Cx32 should enhance self-renewal of CSC to expand the CSC population in HCC.

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