Department of Neurosurgery, Taipei Medical University-Shuang Ho Hospital, Jhongjheng Rd., No.291, New Taipei City, 23561, Taiwan.
Breast Cancer Res. 2011 Oct 24;13(5):R101. doi: 10.1186/bcr3042.
Heat shock proteins (HSPs) are normally induced under environmental stress to serve as chaperones for maintenance of correct protein folding but they are often overexpressed in many cancers, including breast cancer. The expression of Hsp27, an ATP-independent small HSP, is associated with cell migration and drug resistance of breast cancer cells. Breast cancer stem cells (BCSCs) have been identified as a subpopulation of breast cancer cells with markers of CD24-CD44+ or high intracellular aldehyde dehydrogenase activity (ALDH+) and proved to be associated with radiation resistance and metastasis. However, the involvement of Hsp27 in the maintenance of BCSC is largely unknown.
Mitogen-activated protein kinase antibody array and Western blot were used to discover the expression of Hsp27 and its phosphorylation in ALDH + BCSCs. To study the involvement of Hsp27 in BCSC biology, siRNA mediated gene silencing and quercetin treatment were used to inhibit Hsp27 expression and the characters of BCSCs, which include ALDH+ population, mammosphere formation and cell migration, were analyzed simultaneously. The tumorigenicity of breast cancer cells after knockdown of Hsp27 was analyzed by xenograftment assay in NOD/SCID mice. The epithelial-mesenchymal transition (EMT) of breast cancer cells was analyzed by wound-healing assay and Western blot of snail, vimentin and E-cadherin expression. The activation of nuclear factor kappa B (NF-κB) was analyzed by luciferase-based reporter assay and nuclear translocation.
Hsp27 and its phosphorylation were increased in ALDH+ BCSCs in comparison with ALDH- non-BCSCs. Knockdown of Hsp27 in breast cancer cells decreased characters of BCSCs, such as ALDH+ population, mammosphere formation and cell migration. In addition, the in vivo CSC frequency could be diminished in Hsp27 knockdown breast cancer cells. The inhibitory effects could also be observed in cells treated with quercetin, a plant flavonoid inhibitor of Hsp27, and it could be reversed by overexpression of Hsp27. Knockdown of Hsp27 also suppressed EMT signatures, such as decreasing the expression of snail and vimentin and increasing the expression of E-cadherin. Furthermore, knockdown of Hsp27 decreased the nuclear translocation as well as the activity of NF-κB in ALDH + BCSCs, which resulted from increasing expression of IκBα. Restored activation of NF-κB by knockdown of IκBα could reverse the inhibitory effect of Hsp27 siRNA in suppression of ALDH+ cells.
Our data suggest that Hsp27 regulates the EMT process and NF-κB activity to contribute the maintenance of BCSCs. Targeting Hsp27 may be considered as a novel strategy in breast cancer therapy.
热休克蛋白（HSPs）通常在环境压力下诱导产生，作为维持正确蛋白质折叠的伴侣，但它们在许多癌症中经常过表达，包括乳腺癌。Hsp27 是一种 ATP 非依赖性的小 HSP，其表达与乳腺癌细胞的迁移和耐药性有关。乳腺癌干细胞（BCSCs）已被鉴定为具有 CD24-CD44+或高细胞内醛脱氢酶活性（ALDH+）标志物的乳腺癌细胞亚群，并且被证明与辐射抗性和转移有关。然而，Hsp27 在维持 BCSC 中的作用在很大程度上尚不清楚。
采用丝裂原活化蛋白激酶抗体阵列和 Western blot 检测 ALDH+BCSCs 中 Hsp27 的表达及其磷酸化。为了研究 Hsp27 在 BCSC 生物学中的作用，我们使用 siRNA 介导的基因沉默和槲皮素处理来抑制 Hsp27 的表达，并同时分析 BCSC 的特征，包括 ALDH+群体、乳腺球体形成和细胞迁移。通过 NOD/SCID 小鼠异种移植实验分析 Hsp27 敲低后乳腺癌细胞的致瘤性。通过划痕愈合实验和 snail、波形蛋白和 E-钙粘蛋白表达的 Western blot 分析乳腺癌细胞的上皮-间充质转化（EMT）。通过基于荧光素酶的报告基因测定和核转位分析 NF-κB 的激活。
与 ALDH-BCSCs 相比，Hsp27 及其磷酸化在 ALDH+BCSCs 中增加。乳腺癌细胞中 Hsp27 的敲低降低了 BCSC 的特征，如 ALDH+群体、乳腺球体形成和细胞迁移。此外，Hsp27 敲低的乳腺癌细胞中 CSC 频率可减少。用植物类黄酮 Hsp27 抑制剂槲皮素处理细胞也可以观察到抑制作用，而过表达 Hsp27 可以逆转这种抑制作用。Hsp27 的敲低也抑制了 EMT 标志物的表达，如 snail 和波形蛋白的表达降低，E-钙粘蛋白的表达增加。此外，Hsp27 的敲低降低了 ALDH+BCSCs 中 NF-κB 的核转位和活性，这是由于 IκBα表达增加所致。用 IκBα 的 siRNA 敲低恢复 NF-κB 的激活可以逆转 Hsp27 siRNA 抑制 ALDH+细胞的抑制作用。
我们的数据表明，Hsp27 调节 EMT 过程和 NF-κB 活性，有助于维持 BCSC。靶向 Hsp27 可能被认为是乳腺癌治疗的一种新策略。
