Abstract P3-06-04: Notch3 inhibits epithelia-mesenchymal transition by trans-activating GSK3β in breast cancer

Abstract

Abstract Introduction: The Notch and Wnt signaling pathways play critical role in mammary development and tumourigenesis. The epithelia-mesenchymal transition (EMT) is an important process of breast cancer development. Growing evidence shows that Glycogen Synthase Kinase 3 Beta (GSK 3β) is considered as an EMT suppressor of its downregulation of protein snail, a zinc-finger transcriptional repressor that regulates epithelia marker E-cadherin. Previous studies by our group demonstrated that Notch3 may inhibit the emergence and progression of breast cancer. In mammalian cell, GSK3β has been show to physically bind and phosphorylate the intracellular domain of two Notch paralogues. But the relationship between Notch3,GSK3β and EMT in breast cancer still unclear. Intriguingly, we found that knockdown of Notch3 in luminal subtype of breast cancer cells MCF-7 downregulated GSK3β and promoted EMT. In contrast, overexpression of Notch3 in triple-negative cell lines MDA-MB-231 upregulated GSK3βand inhibited EMT. Mechanistically, our results revealed that Notch3 specifically binds to the CSL binding element of the GSK3β promoter and trans-activates its expression. Furthermore, Notch3 and GSK3β expression were positively associated in human breast cancer. Moreover, the mRNA expression of both Notch3 and GSK3βpredicted a better Overall-Survival for breast cancer patients. Our findings demonstrate that Notch3 inhibits breast cancer cells EMT via trans-activating GSK3β expression. Methods: 1. Western blot and/or qRT-PCR were performed to study the expressions of Notch3,GSK3β, β-catenin and EMT related-molecules in breast cancer cell line. 2. Chromatin immunoprecipitation (ChIP) and dual-luciferase assays were carried out to investigate whether Notch3 bound to GSK3β promoter and mediatesGSK3β expression on transcriptional level. 3. Wound healing assay, migration and invasion assays were performed to analyze the effect of Notch3 and GSK3β on breast cancer cell lines migration abilities. 4. The association of Notch3 and GSK3β expression in human breast cancer samples and correlation between Notch3 or GSK3β expression and patient survival in a cohort of human breast cancer patients in Kaplan Meier Plotter database were analyzed. Results: 1. Notch3 up-regulates GSK3β expression in mRNA and protein levels. 2. Notch3 regulates GSK3β expression by binding to the CSL antisense binding site (TTCCCA) positions -308bp to -303bp in the GSK3β promoter. 3. Notch3 intracellular domain (N3ICD) down-regulates the expression of Vimentin, β-catenin, but up-regulates E-cadherin expression. 4. Notch3 inhibits the ability of migration and invasion in breast cancer cell lines. 5. Notch3 and GSK3β expression were positively associated in human breast cancer. The mRNA expression of both Notch3 and GSK3β predicted a better Overall-Survival for breast cancer patients. Summary: We have identified a positive correlation between Notch3 and GSK3β in breast cancer and confirmed a role for the Notch3- GSK3β axis in inhibiting EMT. In addition, we explored in detail and first clarified the possible molecular mechanism of regulating this axis. Our results provide new insights into the complex regulation of breast cancer proliferation and tumorigenesis. We believe that the Notch3- GSK3β axis may be a prospective prognostic predictor for breast cancer. Citation Format: Chen Wei-Ling, Yong-Qu Zhang, Wen-He Huang, Min Chen, Yao-Chen Li, Guo-Jun Zhang. Notch3 inhibits epithelia-mesenchymal transition by trans-activating GSK3β in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-06-04.