Abstract
BackgroundThe 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.MethodsGene 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.ResultsWe 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.ConclusionOur 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.
Highlights
The chemotherapy-resistance of triple-negative breast cancer (TNBC) remains a major challenge
NIMA-related kinase 2 (Nek2) was listed as an TNBC chemotherapy-resistant candidate biomarker by integrated analysis Related gene data set GSE27447 and GSE38959 of TNBC patients who undergoing chemotherapy was downloaded from Gene Expression Omnibus (GEO) database and divided into chemotherapy-sensitive group and
It is found that Nek2B, β-catenin and TCF4 can be binded with the WRE functional area of LEF-1 promoter (Fig. 10a), while they can’t locate in the LEF-1 open reading framework (ORE) (Fig. 10b)
Summary
The chemotherapy-resistance of triple-negative breast cancer (TNBC) remains a major challenge. Because the centrosome cycle is regulated by protein phosphorylation and given the importance of mitotic and centrosomal kinases, they are attractive targets for anti-mitotic anticancer drugs [4]. Nek2B is the only Nek isomer that runs through the whole process of mitosis [5]. It can regulate centrosome aggregation and separation by regulating the mitotic centrosome separation through reversible phosphorylation of its substrates [6, 7]. Nek depletion affected the kinetochores-microtubule attachment, inducing a spindle checkpoint imbalance and abnormal clustering of kinetochore components, resulting in increased sensitivity of cells to the microtubule targeting anticancer drug paclitaxel [9]. In spite of the increasing evidence of the importance of Nek2B in cancer development, its role in cancer is still far from clear
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