Zhongshan Hospital Fudan University Endoscopy Center, Shanghai, 200032, China.
Shanghai Collaborative Innovation Center of Endoscopy, Shanghai, 200032, China.
Cell Commun Signal. 2022 Jun 15;20(1):87. doi: 10.1186/s12964-022-00898-0.
Esophageal Squamous Cell Carcinoma (ESCC) was characterized as a regional-prevalent and aggressive tumor with high morbidity and mortality. NIMA-related kinase 2 (NEK2) is an interesting oncogene, the alteration of which leads to patients-beneficial outcomes. We aimed to explore the role of NEK2 in ESCC and excavate its mechanism.
RNA-seq data were downloaded from TCGA and GEO and analyzed by R software. The protein levels were detected by immunohistochemistry (IHC) or western blot (WB), and mRNA expression was detected by qRT-PCR. The in vitro role of proliferation and migration was detected by Transwell migration assay and by colony formation assay, respectively. The in vivo roles were explored using a subcutaneous xenograft tumor model, where immunofluorescence (IF) and IHC were employed to investigate expression and localization. The interaction between proteins was detected by immunoprecipitation. The stability of proteins was measured by WB in the presence of cycloheximide.
A higher level of NEK2 was found in ESCC than normal esophageal epithelia in GEO, TCGA, and tissue microarray, which was associated with worse prognoses. The NEK2 knockdown impaired the proliferation and migration of ESCC, which also downregulated YAP1 and EMT markers like N-cadherin and Vimentin in vitro. On the contrary, NEK2 overexpression enhanced the migration of ESCC and elevated the levels of YAP1, N-cadherin, and Vimentin. Additionally, the overexpression of YAP1 in NEK2 knocked down ESCCs partly rescued the corresponding decrease in migration. The knockdown of NEK2 played an anti-tumor role in vivo and was accompanied by a lower level and nucleus shuffling of YAP1. In mechanism, NEK2 interacted with YAP1 and increased the stability of both endogenous and exogenous YAP1 by preventing ubiquitination. Moreover, the computer-predicted phosphorylation site of YAP1, Thr-143, reduced the ubiquitination of HA-YAP1, strengthened its stability, and thus influenced the migration in vitro.
NEK2 is a prognostic oncogene highly expressed in ESCC and promotes the progression of ESCC in vitro and in vivo. Mechanistically, NEK2-mediated phosphorylation of YAP1 at Thr-143 protects it from proteasome degradation and might serve as a promising therapeutic target in ESCC. Video Abstract.
食管鳞状细胞癌（ESCC）是一种具有区域性和侵袭性的肿瘤，发病率和死亡率均较高。NIMA 相关激酶 2（NEK2）是一种有趣的癌基因，其改变可使患者受益。我们旨在探讨 NEK2 在 ESCC 中的作用，并挖掘其机制。
从 TCGA 和 GEO 下载 RNA-seq 数据，并使用 R 软件进行分析。通过免疫组织化学（IHC）或蛋白质印迹（WB）检测蛋白质水平，通过 qRT-PCR 检测 mRNA 表达。通过 Transwell 迁移实验和平板克隆形成实验分别检测增殖和迁移的体外作用。通过皮下异种移植肿瘤模型探索体内作用，采用免疫荧光（IF）和免疫组化（IHC）检测表达和定位。通过免疫沉淀检测蛋白质之间的相互作用。在存在环己酰亚胺的情况下通过 WB 测量蛋白质的稳定性。
在 GEO、TCGA 和组织微阵列中，与正常食管上皮相比，ESCC 中发现 NEK2 水平较高，与预后不良相关。NEK2 敲低可削弱 ESCC 的增殖和迁移能力，同时下调体外 YAP1 和 EMT 标志物如 N-钙粘蛋白和波形蛋白。相反，NEK2 过表达增强了 ESCC 的迁移，并提高了 YAP1、N-钙粘蛋白和波形蛋白的水平。此外，在 NEK2 敲低的 ESCC 中转染 YAP1 过表达部分挽救了相应的迁移减少。NEK2 敲低在体内发挥抗肿瘤作用，同时伴有 YAP1 水平降低和核易位。在机制上，NEK2 与 YAP1 相互作用，并通过防止泛素化增加内源性和外源性 YAP1 的稳定性。此外，YAP1 的计算机预测磷酸化位点 Thr-143 减少了 HA-YAP1 的泛素化，增强了其稳定性，并因此影响了体外迁移。
NEK2 是一种在 ESCC 中高度表达的预后癌基因，可促进 ESCC 的体外和体内进展。在机制上，NEK2 介导的 YAP1 的 Thr-143 磷酸化可保护其免受蛋白酶体降解，并可能成为 ESCC 的有前途的治疗靶点。
