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

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.

Keywords: Chemotherapy resistance; Nek2B; Triple-negative breast cancer; Wnt signaling pathway; β-Catenin.

Fig. 1

(a) Venn diagram of the overlaps between GSE27447 DEGs and GSE38959. DEGs. There were 13 common differential genes between the two circles; (b) Relative expression of 13 common differential genes in GSE27447 DEGs and GSE38959 DEGs; (c) Enrichment analysis of the KEGG pathway. *P<0.05; **P<0.01

Fig. 2

Serial slices with immunohistochemical analysis of Nek2B and β-catenin expression in TNBC specimens: Nek2B (a up) and β-catenin (a down) (Normal breast) (200×); Nek2B (b up) and β-catenin (b down) (TNBC (no LNs metastasis) (200×); Nek2B (c up) and β-catenin (c down) (TNBC (LNs metastasis) (200×); Nek2B (d up) and β-catenin (d down) (TNBC (chemotherapy-sensitive) (200×); Nek2B (e up) and β-catenin (e down) (TNBC (chemotherapy-resistant) (200×)

Fig. 3

Kaplan-Meier curves for impact of the expression of Nek2B/β-catenin on disease-free survival (a) and overall survival (b). Log-rank P values are shown above

Fig. 4

(a) CCK8 assay was used to detect the IC50 value of TNBC cell lines; (b) The Nek2B and β-catenin mRNA expression were detected by Q-RT-PCR after chemotherapy-sensitive cells MDA-MB-231 were treated with different concentrations of paclitaxel; (c) Nek2B-siRNA were transfected into drug-resistant cell Hs578T to test drug resistance reversal rate of Nek2B and β-catenin to paclitaxel. *P<0.05; **P<0.01

Fig. 5

The mRNA (a) and protein (b) expression of Nek2B and β-catenin by Q-RT-PCR and Western blot in TNBC cells; Results are represented as mean ± S.E. c The expression pattern of Nek2B and β-catenin were showed by color images of immunofluorescence staining in Hs578T cells. *P<0.05; **P<0.01

Fig. 6

(a) CO-IP was used to detect the combination of endogenous Nek2B and β-catenin in Hs578T cells;(b) Hs578T cells were transfected of HA-β-catenin and FLAG-Nek2B, CO-IP was used to detect the combination of Nek2B and β-catenin

Fig. 7

(a) CO-IP was used to detect whether Nek2B/β-catenin complex participate in the β-catenin phosphorylation and degradation in Hs578T cell; (b) Cytoplasm and nucleus of Hs578 cell line were detected by nucleoplasmic separation assay and CO-IP

Fig. 8

Hs578T (a) and BT20 (b) cells were transfected with Nek2B siRNA, Nek2B and β-catenin mRNA levels were analyzed by qRT-PCR; (c) Western blot analysis was used to detect the Nek2B and p-β-catenin protein expression in Hs578T cells after transfected with Nek2B siRNA. Actin is shown as loading control; MDA-MB-231(d) and MDA-MB-468 (e) cells were transfected with Nek2B plasmid, Nek2B and β-catenin mRNA levels were analyzed by qRT-PCR; (f) Western blot analysis was used to detect the Nek2B and p-β-catenin protein expression in MDA-MB-231 cells after transfected with Nek2B plasmid; Nucleoplasmic separation technique was used to detect the expression of β-catenin in nucleus and cytoplasm after Hs578T cells were transfected with Nek2B siRNA (g) and MDA-MB-231 cells were transfected with Nek2B plasmid (h). *P<0.05; **P<0.01

Fig. 9

Immmunofluorescence was used to show the β-catenin expression after Hs578T cells and BT20 cells were transfected with Nek2B siRNA (a) and MDA-MB-231 cells and 468 cells were transfected with Nek2B plasmid (b)

Fig. 10

The relationship between Nek2B and LEF-l WRE (a) and LEF-l ORF (b) promoter was analysed using CHIP; (c)Hs578T cells were cotransfected with control or Nek2B-siRNA along with the pGL4-LEF-1-Prom-Luc reporter. Data points represent the mean ± S.D. of triplicate samples from two independent experiments. RLU: Relative Luciferase Units; (d) MDA-MB-231 cells were cotransfected with control or Nek2B along with the pGL4-LEF-1-Prom-Luc reporter; (e)Hs578T cells were cotransfected with control or β-catenin-shRNA along with the pGL4-LEF-1-Prom-Luc reporter; (f)MDA-MB-231 cells were cotransfected with control or β-catenin along with the pGL4-LEF-1-Prom-Luc reporter; (g) MDA-MB-231 cells were transfected with Nek2B alone or Nek2B and β-catenin shRNA together along with the pGL4-LEF-1-prom-Luc reporter. Repression of β-catenin could overcome the promotion of LEF-1 promoter activity by Nek2B; ChIP assays was used to detect the combination of β-catenin and LEF-1 promoter with the silence of Nek2B (h), and detect the binding of Nek2B to the LEF-1 promoter with the silence of β-catenin (i). *P<0.05; **P<0.01

Fig. 11

The activity of TOP/FOP was detected after Nek2B-siRNA or Nek2B plasmid were transfected to the cell lines Hs578T and BT20 (a) or MDA-MB-231 and 468 (b), respectively; PCR and westem blot assay were used to detect the expression of downstream target genes of wnt pathway after Nek2B-siRNA was transfected to Hs578T cells (c) and Nek2B plasmid were transfected to MDA-MB-231 cell (d). *P<0.05; **P<0.01

Fig. 12

Nek2B activates the wnt pathway by stabilizing β-catenin. Control, Nek2B, Nek2B + sh-control or Nek2B+ sh-β-catenin plasmids were transfected into MDA-MB-231 cell lines, and the expression levels of TOP/FOP reporting system (a) and the downstream target genes c-myc, cyclinDl and Axin2 of wnt signaling pathway were detected (b) respectively;(c) Transwell cell invasion experiment was used to verify the changes in the invasion ability of MDA-MB-231 cell line after transfection of control, Nek2B, Nek2B + sh-control or Nek2B + sh-β-catenin plasmids. *P<0.05; **P<0.01

Fig. 13

In vivo experiments (a) The nude mice were divided into five groups (1–7) by different treatment, 1: PBS, 2: Nek2B-siRNA control, 3:β-catenin control, 4:paclitaxel, 5: Nek2B siRNA, 6: Nek2B siRNA combining with paclitaxel, 7: Nek2B siRNA and paclitaxel combining with β-catenin. Total RNA extracts from the tumors which were transfacted with Nek2B-siRNA in Hs578T cells (b) and Nek2B plasmid in MDA-MB-231 cells (c) were examined for expression levels of Nek2B, β-catenin and downstream target genes. *P<0.05; **P<0.01

Fig. 14

A hypothesis working model for Nek2B-mediated chemotheropy-resistant mechanism in TNBC. In the classical wnt signaling pathway, the targeted combination of Nek2B and β-catenin can promote the entry of β-catenin into the nucleus and activate the transcription factor TCF/LEF, and then activate the downstream target genes of the wnt pathway and promote drug resistance of TNBC