CKAP4 is a Dickkopf1 receptor and is involved in tumor progression
Hirokazu Kimura, … , Eiichi Morii, Akira Kikuchi
Hirokazu Kimura, … , Eiichi Morii, Akira Kikuchi
Published June 20, 2016
Citation Information: J Clin Invest. 2016;126(7):2689-2705. https://doi.org/10.1172/JCI84658.
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Research Article Cell biology Oncology

CKAP4 is a Dickkopf1 receptor and is involved in tumor progression

Abstract

Dickkopf1 (DKK1) is a secretory protein that antagonizes oncogenic Wnt signaling by binding to the Wnt coreceptor low-density lipoprotein receptor–related protein 6 (LRP6). DKK1 may also regulate its own signaling to promote cancer cell proliferation, but the mechanism is not understood. Here, we identified cytoskeleton-associated protein 4 (CKAP4) as a DKK1 receptor and evaluated CKAP4-mediated DKK1 signaling in cancer cell proliferation. We determined that DKK1 binds CKAP4 and LRP6 with similar affinity but interacts with these 2 receptors with different cysteine-rich domains. DKK1 induced internalization of CKAP4 in a clathrin-dependent manner, further supporting CKAP4 as a receptor for DKK1. DKK1/CKAP4 signaling activated AKT by forming a complex between the proline-rich domain of CKAP4 and the Src homology 3 domain of PI3K, resulting in proliferation of normal cells and cancer cells. Expression of DKK1 and CKAP4 was frequent in tumor lesions of human pancreatic and lung cancers, and simultaneous expression of both proteins in patient tumors was negatively correlated with prognosis and relapse-free survival. An anti-CKAP4 antibody blocked the binding of DKK1 to CKAP4, suppressed AKT activity in a human cancer cell line, and attenuated xenograft tumor formation in immunodeficient mice. Together, our results suggest that CKAP4 is a potential therapeutic target for cancers that express both DKK1 and CKAP4.

Authors

Hirokazu Kimura, Katsumi Fumoto, Kensaku Shojima, Satoshi Nojima, Yoshihito Osugi, Hideo Tomihara, Hidetoshi Eguchi, Yasushi Shintani, Hiroko Endo, Masahiro Inoue, Yuichiro Doki, Meinoshin Okumura, Eiichi Morii, Akira Kikuchi

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Figure 3

DKK1 signaling through CKAP4 activates the PI3K/AKT pathway, resulting in cellular proliferation.

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DKK1 signaling through CKAP4 activates the PI3K/AKT pathway, resulting i...
(A) Control MDCK and MDCK/DKK1-FLAG cells were transfected with siRNA for control or CKAP4, and the cells were then subjected to the 2D cell proliferation assay. Results are shown as the mean ± SD of 3 independent experiments. (B) Control MDCK and MDCK/DKK1-FLAG cells were cultured at densities of 1 × 105 cells in a 35-mm dish 2-dimensionally for 60 hours. The cells were treated for the last 36 hours with 10 μM CHIR99021, 100 μM CKI-7, 10 μM Compound C, 10 μM H-89, 1 μM Y27632, 10 μM U0126, 10 μM SP600125, 10 μM PP2, 200 nM Wortmannin, or 5 μM AKT inhibitor VIII, and then cell numbers were enumerated. Relative cell numbers are shown as fold changes compared with those in DMSO-treated control MDCK cells. Results are shown as the mean ± SD of 3 independent experiments. *P < 0.05; **P < 0.01 (2-tailed Student’s t test). (C) Lysates of control MDCK, MDCK/DKK1-FLAG, or MDCK/DKK1-GPI-FLAG were probed with the indicated antibodies. Clathrin was used as a loading control. (D) Control MDCK or MDCK/DKK1-FLAG cells were transfected with control or CKAP4 siRNA, and cell lysates were probed with the indicated antibodies. Clathrin was used as a loading control. (E) MDCK cells were stimulated with 10 nM DKK1 for the indicated time periods, and cell lysates were probed with the indicated antibodies. (F) Control MDCK or MDCK/DKK1-FLAG cells were treated with 200 nM Wortmannin, 50 μM LY294002, or 5 μM AKT inhibitor VIII for 30 minutes, and cell lysates were probed with the indicated antibodies. (G) Control MDCK or MDCK/DKK1-FLAG cells were transfected with control (scramble) or p110α siRNA, and cell lysates were probed with the indicated antibodies.