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Activation of the Dickkopf1-CKAP4 pathway is associated with poor prognosis of esophageal cancer and anti-CKAP4 antibody may be a new therapeutic drug

Oncogene volume 37, pages 3471–3484 (2018)Cite this article

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Abstract

Aberrant expression of the secretory protein Dickkopf1 (DKK1) is associated with poor prognosis of esophageal squamous cell carcinoma (ESCC), but the underlying mechanism of how DKK1 is involved in aggressiveness of ESCC is not clear. In this study, we show that cytoskeleton-associated protein 4 (CKAP4) functions as a DKK1 receptor in ESCC cells. Immunohistochemical analyses of ESCC revealed that both DKK1 and CKAP4 are minimally expressed in associated normal esophageal squamous mucosa of non-tumor regions, but strongly expressed in tumor lesions. Forty-six of 119 cases (38.7%) were positive for both DKK1 and CKAP4. Those expressing both proteins showed poor prognosis and relapse-free survival. Multivariate analysis demonstrated that expression of both proteins was the poor prognostic factor. The Cancer Genome Atlas data set indicated that the mRNA levels of DKK1 and CKAP4 are significantly elevated in the tumor lesions compared to non-tumor regions. DKK1 bound to CKAP4 at endogenous levels. DKK1 induced the internalization of CKAP4 from and its recycling to the plasma membrane. AKT was activated in ESCC cells in which DKK1 was highly expressed and CKAP4 was localized to the plasma membrane. Knockdown of either DKK1 or CKAP4 inhibited AKT activity and cell proliferation in vitro and xenograft tumor formation. Wild-type CKAP4 or DKK1, but not a DKK1 mutant that was unable to bind to CKAP4, rescued phenotypes induced by CKAP4 or DKK1 knockdown, respectively. The anti-CKAP4 antibody also inhibited AKT activity and suppressed xenograft tumor formation. In contrast, in ESCC cells in which DKK1 was marginally expressed, knockdown of CKAP4 or anti-CKAP4 antibody affected neither AKT activity nor cell proliferation. These findings suggest that the DKK1-CKAP4 pathway promotes ESCC cell proliferation and that CKAP4 might represent a novel therapeutic target for ESCCs expressing both DKK1 and CKAP4.

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Acknowledgements

We thank Drs. Yuri Terao and Yuka Umeki at the Center for Medical Research and Education and Department of Molecular Biology and Biochemistry, respectively, Graduate School of Medicine, Osaka University for the technical assistance for the preparation of MISSION TRC shRNAs and mRNA, and qPCR, and Western blotting. This work was supported by Grants-in-Aid for Scientific Research (2013–2015) (No. 25250018) and to AK (2016–2017) (No. 16H06374) to AK, Grants-in-Aid for Scientific Research on Innovative Areas (2011–2015) (No. 23112004) to AK, Grants-in-Aid for Young Scientists (Start-up) (2016–2017) (No. 16H06944) to HK, and Grant-in-Aid for Young Scientists (B) (2015–2017) (No. T15K198870) to NS from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and also supported by the Project Promoting Support for Drug Discovery to KF and the Project for Cancer Research And Therapeutic Evolution (P-CREATE) to AK from the Japan Agency for Medical Research and development, AMED, and by grants to AK from the Yasuda Memorial Foundation.

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  1. These authors contributed equally: Naoki Shinno, Hirokazu Kimura.

Authors and Affiliations

  1. Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, 565-0871, Japan

    Naoki Shinno, Shuji Takiguchi, Masaki Mori & Yuichiro Doki

  2. Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, 565-0871, Japan

    Hirokazu Kimura, Ryota Sada, Katsumi Fumoto & Akira Kikuchi

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Correspondence to Akira Kikuchi.

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Shinno, N., Kimura, H., Sada, R. et al. Activation of the Dickkopf1-CKAP4 pathway is associated with poor prognosis of esophageal cancer and anti-CKAP4 antibody may be a new therapeutic drug. Oncogene 37, 3471–3484 (2018). https://doi.org/10.1038/s41388-018-0179-2

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