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Downregulated ATP6V1B1 expression acidifies the intracellular environment of cancer cells leading to resistance to antibody-dependent cellular cytotoxicity

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Abstract

Among several mechanisms for the resistance of human epidermal growth factor receptor 2-overexpressing (HER2 +) cancer cells to trastuzumab, little is known regarding the mechanism underlying the resistance to trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC). Cell death due to ADCC is caused by apoptosis of target cells induced by granzymes released from natural killer cells. Because optimal granzyme physiological activity occurs at neutral pH, we assumed that the pH of the intracellular environment influences the cytotoxic effects of granzymes. We established ADCC-resistant cells and compared them with wild-type cells in terms of the expression of intracellular pH-regulating genes. The expression of ATP6V1B1, which encodes a component of vacuolar ATPases, was downregulated in the ADCC-resistant cells. Thus, to functionally characterize ATP6V1B1, we used a CRISPR/Cas9 system to generate ATP6V1B1-knockout SKBR3 and JIMT-1 cells (both HER2 + human breast cancer cell line). The resulting cells exhibited significantly less ADCC than the control SKBR3 and JIMT-1 cells. The intracellular pH of the ATP6V1B1-knockout SKBR3 and JIMT-1 cells was significantly lower than control SKBR3 and JIMT-1cells. An analysis of granzyme dynamics during the ADCC reaction in cancer cells revealed that granzymes degraded intracellularly in the control SKBR3 and JIMT-1 cells and accumulated in ATP6V1B1-knockout cells, but were not cytotoxic. These findings suggest that decreased vacuolar ATPase activity alters the cytoplasmic pH of cancer cells to create an environment that is less suitable for granzyme bioactivity, which adversely affects the induction of apoptosis of cancer cells by NK cells.

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Acknowledgements

This research was supported by a grant from the Japan Society for the Promotion of Science through the KAKENHI Grant Numbers 24591901. We thank Keiko Sakamoto, Yuji Fukushima, Masashi Inoue and Sunao Tanaka for teaching experimental techniques and helping with the experiments.

Funding

This research was supported by a grant from the Japan Society for the Promotion of Science (KAKENHI Grant Number 24591901).

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Authors and Affiliations

  1. Department of Breast Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Mariko Nishie, Eiji Suzuki, Kosuke Kawaguch, Fengling Pu, Takeshi Kotake, Moe Tsuda, Ayane Yamaguchi & Masakazu Toi

  2. Department of Immunosenescence, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Masakazu Hattori

  3. Department of Diagnostic Pathology, Kyoto University Hospital, Kyoto, Japan

    Tatsuki R. Kataoka & Masahiro Hirata

  4. Department of Breast Surgery, Kansai Medical University, Osaka, Japan

    Tomoharu Sugie

  5. Department of Breast Surgery, Kyoto University Hospital, 54 Shogoin-kawaharacho, Sakyo-ku, Kyoto, 606-8507, Japan

    Eiji Suzuki

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  1. Mariko Nishie
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Correspondence to Eiji Suzuki.

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Nishie, M., Suzuki, E., Hattori, M. et al. Downregulated ATP6V1B1 expression acidifies the intracellular environment of cancer cells leading to resistance to antibody-dependent cellular cytotoxicity. Cancer Immunol Immunother 70, 817–830 (2021). https://doi.org/10.1007/s00262-020-02732-3

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  • DOI: https://doi.org/10.1007/s00262-020-02732-3

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