Nek2B 通过稳定β-catenin 激活 wnt 通路并促进三阴性乳腺癌化疗耐药。

Nek2B activates the wnt pathway and promotes triple-negative breast cancer chemothezrapy-resistance by stabilizing β-catenin.

机构信息

Department of pathology, The Second Clinical Medical College, Shanxi Medical University, 382 Wuyi Road, Taiyuan, 030001, People's Republic of China.

Department of general surgery, The Second Clinical Medical College, Shanxi Medical University, 382 Wuyi Road, Taiyuan, 030001, People's Republic of China.

出版信息

J Exp Clin Cancer Res. 2019 Jun 7;38(1):243. doi: 10.1186/s13046-019-1231-y.

DOI:10.1186/s13046-019-1231-y

PMID:31174562

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6556028/

Abstract

BACKGROUND

The chemotherapy-resistance of triple-negative breast cancer (TNBC) remains a major challenge. The Nek2B kinase and β-catenin serve as crucial regulators of mitotic processes. The aim of this study was to test the correlation between Nek2B and TNBC chemotherapy sensitivity, and to determine the regulation of Nek2B on β-catenin and wnt/β-catenin signal pathway.

METHODS

Gene Expression Omnibus(GEO) databases were used to gather gene exprsssion data of TNBC patients who undergoing chemotherapy. The co-expression of Nek2B and β-catenin in TNBC surgical sections and cells were analysed by immunohistochemistry, Q-RT-PCR, Western-blot and immunofluorescent staining. The impact of the expression of Nek2B and β-catenin in prognosis was also assessed using the Kaplan-Meier curves. CCK8 assay was used to detect the IC50 value of TNBC cell line. The endogenous binding capacity of Nek2B and β-catenin and phosphorylation of β-catenin by Nek2B were detected using co-immunoprecipitation (CO-IP). Chromatin immune-precipitation (ChIP) analysis and Luciferase Assays were used to evaluate the binding ability of the Nek2B, β-catenin and TCF4 complex with LEF-1 promoter. Nek2B-siRNA and Nek2B plasmid were injected into nude mice, and tumorigenesis was monitored.

RESULTS

We found that overexpression of Nek2B and β-catenin in TNBC samples, was associated with patients poor prognosis. Patients with positive Nek2B expression were less sensitive to paclitaxel-containing neoadjuvant chemotherapy. Interestingly, in a panel of established TNBC cell line, Nek2B and β-catenin were highly expressed in cells exhibiting paclitaxel resistance. Our data also suggest that β-catenin binded to and was phosphorylated by Nek2B, and was in a complex with TCF4. Nek2B mainly regulates the expression of β-catenin in TNBC nucleus. Nek2B, β-catenin and TCF4 can be binded with the WRE functional area of LEF-1 promoter. Nek2B can activite wnt signaling pathway and wnt downstream target genes. The tumors treated by Nek2B siRNA associated with paclitaxel were the smallest in nude mouse, and Nek2B can regulate the expression of β-catenin and wnt downstream target genes in vivo.

CONCLUSION

Our study suggested that Nek2B can bind to β-catenin and the co-expression correlated with TNBC patients poor prognosis. It appears that Nek2B and β-catenin might synergize to promote chemotherapy resistance.

摘要

背景

三阴性乳腺癌（TNBC）的化疗耐药性仍是一个主要挑战。Nek2B 激酶和 β-连环蛋白作为有丝分裂过程的关键调节剂。本研究旨在检验 Nek2B 与 TNBC 化疗敏感性的相关性，并确定 Nek2B 对β-连环蛋白和 wnt/β-连环蛋白信号通路的调控作用。

方法

使用基因表达综合数据库（GEO）收集接受化疗的 TNBC 患者的基因表达数据。通过免疫组化、Q-RT-PCR、Western-blot 和免疫荧光染色分析 TNBC 手术标本和细胞中 Nek2B 和 β-连环蛋白的共表达。还使用 Kaplan-Meier 曲线评估 Nek2B 和 β-连环蛋白表达对预后的影响。使用 CCK8 测定法检测 TNBC 细胞系的 IC50 值。使用免疫共沉淀（CO-IP）检测 Nek2B 和 β-连环蛋白的内源性结合能力以及 Nek2B 对 β-连环蛋白的磷酸化。使用染色质免疫沉淀（ChIP）分析和荧光素酶测定评估 Nek2B、β-连环蛋白和 TCF4 复合物与 LEF-1 启动子的结合能力。将 Nek2B-siRNA 和 Nek2B 质粒注入裸鼠，监测肿瘤发生情况。

结果

我们发现，TNBC 样本中 Nek2B 和 β-连环蛋白的过度表达与患者预后不良有关。表达 Nek2B 的患者对含紫杉醇的新辅助化疗不敏感。有趣的是，在一组已建立的 TNBC 细胞系中，紫杉醇耐药细胞中高度表达 Nek2B 和 β-连环蛋白。我们的数据还表明，β-连环蛋白与 Nek2B 结合并被 Nek2B 磷酸化，并与 TCF4 形成复合物。Nek2B 主要调节 TNBC 核内的 β-连环蛋白表达。Nek2B、β-连环蛋白和 TCF4 可以与 LEF-1 启动子的 WRE 功能区结合。Nek2B 可以激活 wnt 信号通路和 wnt 下游靶基因。用 Nek2B siRNA 处理的肿瘤在裸鼠中最小，Nek2B 可以在体内调节 β-连环蛋白和 wnt 下游靶基因的表达。