Nestin positively regulates the Wnt/β-catenin pathway and the proliferation, survival and invasiveness of breast cancer stem cells.
Nestin positively regulates the Wnt/β-catenin pathway and the proliferation, survival and invasiveness of breast cancer stem cells.
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DOI：
10.1186/s13058-014-0408-8
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发表时间：
2014-07-24
期刊：
影响因子：
--
通讯作者：
Liu C
中科院分区：
文献类型：
--
作者：
Zhao Z;Lu P;Zhang H;Xu H;Gao N;Li M;Liu C
我们研究了巢蛋白在三阴性乳腺癌中的表达，并研究了巢蛋白表达的调节如何影响体外乳腺癌干细胞（CSC）的细胞周期进展、存活、侵袭和调控信号。采用免疫组化方法检测150例三阴性乳腺癌组织中巢蛋白的表达。通过测定来自12个乳腺癌组织的天然存在的Nestinhigh/Nestinlow CSC以及来自26个临床标本的CSC来检查Nestin表达在肿瘤发生中的作用，其中通过遗传操作实现Nestin过表达和沉默，以确定它们形成乳腺球和诱导实体瘤的能力。流式细胞术和transwell法检测Nestin沉默的乳腺CSC的细胞周期进程、自发凋亡和侵袭力。Western blotting法检测EMT和Wnt/β-catenin通路相关分子的相对表达水平。巢蛋白表达与三阴性乳腺癌患者的生存率显著相关（P = 0.01）。Nestinhigh乳腺CSC在体外快速形成典型的乳腺球。Nestinhigh，而不是Nestinlow CSC，在体内有效地形成实体瘤。巢蛋白沉默诱导细胞周期停滞在G2/M期（52.03%对对照组的19.99%），并促进细胞凋亡（36.45%对对照组的8.29%）。巢蛋白沉默还抑制乳腺CSC的侵袭性，并且与显著上调的E-钙粘蛋白相关，而N-钙粘蛋白、波形蛋白、α-平滑肌肌动蛋白（α-SMA）、基质金属蛋白酶-2（MMP-2）、MMP-9和血管内皮生长因子（VEGF）表达下调（均P <0.05）。巢蛋白沉默还上调CSC中Axin、糖原合成酶激酶-3 β（GSK-3β）、腺瘤性结肠息肉病（APC）和过氧化物酶体增殖物激活受体α（PPARa）的表达，并下调CSC中β-连环蛋白、c-Myc、细胞周期蛋白D和MMP-7的表达。抑制Wnt/β-catenin途径可减轻Nestinhigh CSC中乳腺球的形成，而抑制GSK-3β可促进Nestinlow CSC中乳腺球的形成（均P <0.05）。我们的数据表明Nestin通过增强Wnt/β-catenin激活而正向调节乳腺CSC的增殖、存活和侵袭性。本文的在线版本（doi：10.1186/s13058-014-0408-8）包含补充材料，可供授权用户使用。
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 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. Nestin 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). Our data indicates that Nestin positively regulates the proliferation, survival and invasiveness of breast CSC by enhancing Wnt/β-catenin activation. The online version of this article (doi:10.1186/s13058-014-0408-8) contains supplementary material, which is available to authorized users.
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