The Notch receptor is a highly conserved type I transmembrane glycoprotein that is involved in cell differentiation, proliferation, and survival, which play important roles in various tumors . Notch1–4 has a highly similar structure and corresponding functional role in mammals, but has a distinct function in normal breast development and breast cancer . It has been identified that inhibition of Notch1 expression can reverse the EMT process of breast cancer cells, thereby inhibiting cell migration and invasion . Furthermore, a clinical study found that Notch1 correlated to a poor prognosis in patients with breast cancer . Notch2 overexpression predicts better overall survival in women with breast cancer, which is completely different from Notch1 . A recent study showed the carcinogenic effect of Notch4. Zhou et al. revealed that Notch4 overexpression can promote the EMT of breast cancer cells and maintain the self-renewal properties of mesenchymal breast cancer stem cells , which is of concern and is associated with endocrine drug resistance in breast cancer.
Although the basic structure of Notch3 bears a resemblance to other Notch receptor members, many differences exist in the intracellular domain as well as a special shorter transcription activation domain. Several studies have indicated that Notch3 may play a considerable role in the formation of mammary epithelial cells during breast development . Daniel et al.  revealed that Notch3 could label luminal progenitor cells and inhibit the proliferation of tumor cells. Cui et al.  found that Notch3 regulated cell senescence by regulating P21, thus inhibiting the occurrence and development of tumors. Similarly, Notch3 is associated with the inhibition of cell proliferation and apoptosis in HER2-negative breast cancer . Our previous studies revealed that ERα inhibits EMT by suppressing Bmi1 , and Notch3 upregulates ERα . Meanwhile, another study from our group showed that Notch3 can inhibit EMT in breast cancer epithelial cells by transactivating Kibra . Therefore, Notch3 may have a pivotal role in tumor suppression, especially in breast cancer EMT.
The loss of E-cadherin expression is regarded as an essential event in EMT. It leads to disruption of epithelial cell polarity induced EMT  thus maintaining the mesenchymal phenotype and enhancing the invasion and metastasis of cancer cells . A variety of molecular mechanisms can repress E-cadherin expression via transcriptional inhibition and promotor methylation. Doble et al. demonstrated that GSK3β acts as an EMT suppressor of the zinc-finger transcriptional repressor, snail, which regulates epithelial marker E-cadherin . These results indicated that both Notch3 and GSK3β inhibit EMT in breast cancer and GSK3β can physically bind and phosphorylate the intracellular domain of two Notch paralogs [18, 19]. However, the interaction of Notch3 and GSK3β in EMT, a potential key step in breast cancer metastasis that contributes to breast cancer-related deaths, remains unclear.
Our results revealed that GSK3β was highly expressed in the luminal-type breast cancer cell lines, MCF-7 and T-47D, but was expressed at low levels in the TNBC cell lines, MDA-MB-231 and BT-549. This is consistent with our previous findings that the expression pattern of Notch3 is similar to GSK3β in these cell lines. In addition, our previous studies found that the CSL-binding element, GGGAA, participated in regulating the ER and GATA3 [21, 39]. In this study, we demonstrated that Notch3 bound to the GSK3β promoter, which contains CSL-binding elements. Here, we found that the luciferase reporter activity of GSK3β decreased in the form of a concentration gradient after Notch3 knockdown in MCF-7 cells. In contrast, GSK3β-mediated luciferase activity increased after N3ICD over-expression in MDA-MB-231 cells. Our study on its molecular mechanism revealed that inhibition of Notch3 downregulated GSK3β mRNA and protein levels in MCF-7 cells significantly. Conversely, Notch3ICD overexpression upregulated that of GSK3β in MDA-MB-231 cells as illustrated in Figure 7.
In summary, our research findings indicated that Notch3 represses the processes of EMT in breast cancer, which is consistent with our previous reports. Remarkably, this finding demonstrates for the first time that Notch3 may inhibit the EMT process in breast cancer cells through transcriptionally up-regulating GSK3β. From data obtained from clinical tissue, Notch3 expression was significantly consistent with GSK3β expression in breast cancer tissue samples. In a prognostic analysis, high expression of mRNA of both Notch3 and GSK3β was related to better RFS in all patients with breast cancer studied, which implies that Notch3 and GSK3β are beneficial biomarkers in this disease. Our results highlight a novel mechanism for exploring how Notch3 regulates EMT as well as the crosstalk between Notch and Wnt signaling pathways. This may have important implications for identifying new biomarkers for the prognosis of and as therapeutic targets in breast cancer.