Nestin positively regulates the Wnt/β-catenin pathway and the proliferation, survival and invasiveness of breast cancer stem cells

Introduction: We investigated Nestin expression in triple-negative breast cancer and examined how the modulation of Nestin expression affects cell cycle progression, survival, invasion and regulatory signaling in breast cancer stem cells (CSC) in vitro. Methods: Nestin expression in 150 triple-negative breast cancer specimens were examined by immunohistochemistry. The role of Nestin expression in tumorigenesis was examined by assaying naturally occurring Nestin(high)/Nestin(low) CSC from 12 breast cancer tissues, as well as CSC from 26 clinical specimens, where Nestin overexpression and silencing was achieved by genetic manipulation, for their ability to form mammospheres and induce solid tumors. Cell cycle progression, spontaneous apoptosis and invasiveness of Nestin-silenced breast CSC were investigated by flow cytometry and transwell assays. The relative levels of expression of epithelial-mesenchymal transition (EMT) and Wnt/beta-catenin pathway-related molecules were determined by western blotting. Results: Nestin expression was significantly associated with poor survival in patients with triple-negative breast cancer (P = 0.01). Nestin(high) breast CSC rapidly formed typical mammospheres in vitro. Nestinhigh, but not Nestin(low) CSC, efficiently formed solid tumors in vivo. Nestin silencing induced cell cycle arrest at G2/M (52.03% versus 19.99% in controls) and promoted apoptosis (36.45% versus 8.29% in controls). Nestin silencing also inhibited breast CSC invasiveness, and was associated with significantly upregulated E-cadherin, while N-cadherin, vimentin, a-smooth muscle actin (a-SMA), matrix metalloproteinase-2 (MMP-2), MMP-9 and vascular endothelial growth factor (VEGF) expression was downregulated (P < 0.05 for all). Nestin silencing also upregulated Axin, glycogen synthase kinase-3 beta (GSK-3 beta), adenomatous polyposis coli (APC), and peroxisome proliferator-activated receptor alpha (PPARa), and downregulated beta-catenin, c Myc, cyclin D and MMP 7 expression in CSC. Inhibition of the Wnt/beta-catenin pathway mitigated mammosphere formation in Nestinhigh CSC, while inhibition of GSK-3 beta promoted the mammosphere formation in Nestinlow CSC (P < 0.05 for all). Conclusions: Our data indicates that Nestin positively regulates the proliferation, survival and invasiveness of breast CSC by enhancing Wnt/beta-catenin activation.