Abstract
IntroductionWe 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.MethodsNestin 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 Nestinhigh/Nestinlow 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/β-catenin pathway-related molecules were determined by western blotting.ResultsNestin expression was significantly associated with poor survival in patients with triple-negative breast cancer (P = 0.01). Nestinhigh breast CSC rapidly formed typical mammospheres in vitro. Nestinhigh, but not Nestinlow 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-3β), adenomatous polyposis coli (APC), and peroxisome proliferator-activated receptor alpha (PPARa), and downregulated β-catenin, c-Myc, cyclin D and MMP-7 expression in CSC. Inhibition of the Wnt/β-catenin pathway mitigated mammosphere formation in Nestinhigh CSC, while inhibition of GSK-3β promoted the mammosphere formation in Nestinlow CSC (P <0.05 for all).ConclusionsOur data indicates that Nestin positively regulates the proliferation, survival and invasiveness of breast CSC by enhancing Wnt/β-catenin activation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0408-8) contains supplementary material, which is available to authorized users.
Highlights
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
Nestin expression is associated with poor survival in patients with triple-negative breast cancer Nestin is an intermediate filament protein expressed by neural precursors, muscle, and stem cells [34], and has been shown to regulate cell proliferation
Our previous study showed that Nestin is expressed in human breast cancer tissue, and that its expression was associated with lymph node metastasis in a cohort of breast cancer patients [16]
Summary
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. Patients with ‘triple-negative’ breast cancer, referring to absence in the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), are insensitive to hormonal therapy or HER2targeted agents [1]. These patients are at increased risk for relapse and metastasis of breast cancer, which is often incurable and a leading cause of female mortality [2,3,4]. There is currently no effective therapy to control the recurrence and metastasis of triple-negative breast cancer. Understanding the molecular mechanisms underlying the recurrence and metastasis of triple-negative breast cancer may reveal new therapeutic targets, and can significantly improve the management of patients with triple-negative breast cancer
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