Notch3 Transactivates Glycogen Synthase Kinase-3-Beta and Inhibits Epithelial-to-Mesenchymal Transition in Breast Cancer Cells

Abstract

As a critical transformational process in the attributes of epithelial cells, epithelial-to-mesenchymal transition (EMT) is involved in tumor invasion, metastasis, and resistance to treatment, which contributes to the ultimate death of some patients with breast cancer. Glycogen synthase kinase-3-beta (GSK3β) is thought to be an EMT suppressor that down-regulates the protein, snail, a zinc finger transcription inhibitor, and regulates E-cadherin expression and the Wnt signaling pathway. Our previous studies have shown that Notch3 also inhibits EMT in breast cancer. In mammary gland cells, GSK3β physically bound and phosphorylated the intracellular domain of two Notch paralogs: N1ICD was positively regulated, but N2ICD was negatively regulated; however, the relationship between Notch3, GSK3β, and EMT in breast cancer is still unclear and crosstalk between Notch3 and GSK3β has not been widely investigated. In this study, we revealed that Notch3 was an essential antagonist of EMT in breast cancer cells by transcriptionally upregulating GSK3β. In breast cancer, MCF-7 and MDA-MB-231 cell lines, the silencing of Notch3 reduced GSK3β expression, which is sufficient to induce EMT. Conversely, ectopic Notch3 expression re-activated GSK3β and E-cadherin. Mechanistically, Notch3 can bind to the GSK3β promoter directly and activate GSK3β transcription. In human breast cancer samples, Notch3 expression is positively associated with GSK3β (r = 0.416, p = 0.001); moreover, high expressions of Notch3 and GSK3β mRNA are correlated to better relapse-free survival in all breast cancer patients via analysis in "the Kaplan-Meier plotter" database. In summary, our preliminary results suggested that Notch3 might inhibit EMT by trans-activating GSK3β in breast cancer cells. The suppression of Notch3 expression may contribute to EMT by transcriptionally downregulating GSK3β in breast cancer.

Keywords: GSK3β; Notch3; breast cancer; epithelial-to-mesenchymal transition; prognosis.

Figure 1

Notch3 is expressed in the luminal subtype and modulates GSK3β expression in breast cancer cell lines. (A): Heat map representing the correlation of DNA microarrays of GSK3β and Notch1–4, which was obtained from Breast Cancer Gene−Expression Miner v4.7. (B): A significant positive correlation existed between Notch3 and GSK3β. The correlation coefficient was r = 0.15, p < 0.0001. (C): Notch3 and GSK3β expression in distinct subtypes of breast cancer cell lines detected by western blotting. (D): Expression of Notch3 and GSK3β mRNA analyzed by RT–PCR in breast cancer cell lines. (E): Immunofluorescence staining of GSK3β and Notch3 in MCF-7 and T-47D cells. Nuclei were counterstained with DAPI. The scale bar represents 50 μm. ** p < 0.01.

Figure 2

Ectopic Notch3 induces GSK3β expression and inhibits epithelial–mesenchymal transition (A): MCF-7 cells were transiently transfected with small interfering (si) RNA of Notch3 or vehicle (control). After 48 h, quantitative reverse transcription (qRT)–PCR was used to assess the mRNA levels of Notch3 and GSK3β. (B,C): Protein levels of Notch3, E-cadherin, and vimentin in MCF-7 cells transfected with siRNA-negative control (NC) or siRNA-Notch3 after 48 h were measured by western blotting. (D): MDA-MB-231cells were transiently transfected with pCLE-Notch 3 intracellular domain (N3ICD) or pCLE vehicle (control). After 48 h, qRT–PCR was used to assess the mRNA levels of Notch3 and GSK3β. (E,F): Protein levels of Notch3, E-cadherin, and vimentin in MDA-MB-231 cells transfected with pCLE-N3ICD or vehicle (control) after 48 h were measured by western blotting. (G): Immunofluorescence staining of GSK3β and Notch3 in MCF-7 and T-47D cells treated with control siRNA or siNotch3. Nuclei were counterstained with DAPI. The scale bar represents 50 μm.* p < 0.05, *** p < 0.001.

Figure 3

Notch3 transactivates GSK3β by directly binding to the GSK3β promoter. (A): A schema of the four CSL-binding element-containing primers (GSK3β 1−1, GSK3β 2, and GSK3β 3 regions containing respective single CSL-binding elements; region GSK3β 1−2 containing two CSL-binding elements) used for chromatin immunoprecipitation (ChIP) assays. (B): Products of ChIP were amplified by PCR and analyzed by 2% agarose gel electrophoresis. A specific band was seen in the GSK3β 2 region; (C): MCF-7 cells co-transfected with gradient concentrations of siRNA-Notch3 (“+” 10 pmol, “++” 20 pmol, “+++” 40 pmol, “−” 0 pmol) or the same concentrations of siRNA-negative control (NC) (“+” 20 pmol, “++” 30 pmol,“+++” 40 pmol,“−” 0 pmol), 10 ng pRL-SV40, and 200 ng pGL3-GSK3β-enhancer. (D): MDA-MB-231 cells co-transfected with gradient concentrations of pCMV- N3ICD; “+” 200 ng, “++” 400 ng, “+++” 800 ng, “−” 0 ng) or the same concentrations of pCMV (“+” 400 ng, “++” 600 ng, “+++” 800 ng, “−” 0 ng), 10 ng pRL-SV40, and 200 ng pGL3-GSK3β-enhancer. * p < 0.05, ** p < 0.01.

Figure 4

Restoration of GSK3β expression reverses Notch3-mediated suppression of migration and invasion in breast cancer cells. (A): Notch3 inhibition induced MCF-7 cell growth in vitro; this effect was attenuated by GSK3β overexpression. (B): Quantitative analysis of the wound healing rate of cells in each treatment group MCF-7siRNA-NC, MCF-7siRNA-Notch3 and MCF-7siRNA-Notch3 + GSK3β. (C,D): MCF-7siRNA-Notch3, MCF-7siRNA-NC, and MCF-7siRNA-Notch3 + pCMV3-GSK3β-GFP-Spark cells were subjected to transwell migration and invasion analysis. The mean ± SD of migrated cells of three independent experiments is shown in the panel. (E): N3ICD overexpression inhibited MDA-MB-231 cell growth in vitro; this effect was attenuated by GSK3β knockdown. (F): Quantitative analysis of the wound healing rate of cells in each treatment group above. (G,H): MDA-MB-231pCMV, MDA-MB-231pCMV-N3ICD, and MDA-MB-231pCMV-N3ICD + psi-U6.1/eGFP/shRNA-GSK3β cells were subjected to transwell migration and invasion analysis. The mean ± SD of migrated cells of three independent experiments is shown in the panel. Magnification 400×. * p < 0.05. GFP, green fluorescent protein; NC, negative control; siRNA, small interfering RNA.

Figure 5

The correlation between Notch3 and GSK3β expression in human breast cancer specimens. (A): Representative image of Notch3 negative (−) staining cells in human breast cancer tissue. (B): Representative image of Notch3 positive (+) staining cells. (C): Representative image of GSK3β negative (−) staining cells. (D): Representative image of GSK3β positive (+) staining cells. Magnification 200×.

Figure 6

Analysis of Notch3 and GSK3β expression of RFS in patients with breast cancer. (A,D,G,M): High expression of Notch3 resulted in better recurrence-free survival (RFS) among different breast cancer subtypes but not the basal-like subtype (J). (B,E,H,N): High expression of GSK3β resulted in a better RFS among different breast cancer subtypes but not the basal-like subtype (K). A superior RFS was observed for breast cancer patients expressing both high Notch3 and GSK3β levels in all patients, luminal A subtype (C,F) but not luminal B, basal-like, or Her 2 subtypes (I,L,O).