Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, 565-0871, Japan.
Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, 565-0871, Japan.
Oncogene. 2018 Jun;37(26):3471-3484. doi: 10.1038/s41388-018-0179-2. Epub 2018 Mar 22.
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.
分泌蛋白 Dickkopf1（DKK1）的异常表达与食管鳞状细胞癌（ESCC）的预后不良相关，但 DKK1 如何参与 ESCC 的侵袭性的潜在机制尚不清楚。在这项研究中，我们表明细胞骨架相关蛋白 4（CKAP4）在 ESCC 细胞中作为 DKK1 的受体发挥作用。对 ESCC 的免疫组织化学分析显示，DKK1 和 CKAP4 在非肿瘤区域相关的正常食管鳞状粘膜中均低表达，但在肿瘤病变中强烈表达。在 119 例病例中有 46 例（38.7％）同时表达 DKK1 和 CKAP4。表达两种蛋白的患者预后不良且无复发生存率低。多变量分析表明，两种蛋白的表达是预后不良的因素。癌症基因组图谱数据集表明，与非肿瘤区域相比，肿瘤病变中 DKK1 和 CKAP4 的 mRNA 水平显着升高。DKK1 在内源性水平上与 CKAP4 结合。DKK1 诱导 CKAP4 从内体内化并循环到质膜。在 DKK1 高表达且 CKAP4 定位于质膜的 ESCC 细胞中，AKT 被激活。体外和异种移植肿瘤形成中，敲低 DKK1 或 CKAP4 均可抑制 AKT 活性和细胞增殖。野生型 CKAP4 或 DKK1，但不是不能与 CKAP4 结合的 DKK1 突变体，分别挽救了由 CKAP4 或 DKK1 敲低诱导的表型。抗 CKAP4 抗体也抑制 AKT 活性并抑制异种移植肿瘤形成。相比之下，在 DKK1 低表达的 ESCC 细胞中，敲低 CKAP4 或抗 CKAP4 抗体均不影响 AKT 活性或细胞增殖。这些发现表明 DKK1-CKAP4 途径促进 ESCC 细胞增殖，并且 CKAP4 可能是同时表达 DKK1 和 CKAP4 的 ESCC 的新型治疗靶标。
