Hsp27 participates in the maintenance of breast cancer stem cells

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Introduction$ J" m; s2 H" L) d+ H7 Y5 T
Heat shock proteins (HSPs) are normally induced under environmental stress to serve as chaperone 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.0 G6 B0 H  W- w0 X2 P' z- X% Z

6 R% ^- d, g! h0 @* BMethods
( N% I5 z% Z2 k: i& c) oMitogen-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-kappa B) was analyzed by luciferase-based reporter assay and nuclear translocation.6 Y/ |2 {. w: ~0 v( r# C: X

( R% I& Y+ A' g5 b7 G7 YResults, ?$ b( U. z8 C' J2 `& i; x2 a
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-kappa B in ALDH+ BCSCs which resulted from increasing expression of Ikappa B alpha. Restored activation of NF-kappa B by knockdown of I kappa B alpha could reverse the inhibitory effect of Hsp27 siRNA in suppression of ALDH+ cells.
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Conclusions
/ b6 z& H" E$ L7 _4 H* W  xOur data suggest that Hsp27 regulates EMT process and NF-kappa B activity to contribute the maintenance of BCSCs. Targeting Hsp27 may be considered as a novel strategy in breast cancer therapy.+ F3 B6 e. y  ?! q1 K

0 [. d. {6 m; i( Lhttp://breast-cancer-research.com/content/13/5/R101/abstract

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" ?7 _/ R6 V! D, r! s7 lHsp27，一种不依赖于ATP的小分子热休克蛋白，与乳腺癌细胞的细胞迁移和抗药物治疗相关联。人们已经鉴定出乳腺癌干细胞(breast cancer stem cells, BCSCs)是乳腺癌细胞的一个亚群，带有CD24-CD44+ 分子标记物或具有较高的细胞内乙醛脱氢酶活性(aldehyde dehydrogenase activity, ALDH+)，能够跟癌症抵抗放射性治疗和转移相关联。但是Hsp27是否参与BCSCs的维持仍然很不甚明了。
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' K% q% o% U3 e, n9 S; C# ^在该研究中，研究人员发现，Hsp27表达水平及其磷酸化相比于ALDH-的非BCSCs细胞增加不少。在乳腺癌细胞中，敲除Hsp27会减弱BCSCs的特性，比如ALDH+细胞数量，乳腺球(mammosphere)形成和细胞迁移，而且体内乳腺癌癌干细胞也能够减少。当用Hsp27的一种黄酮类抑制物槲皮苷(quercetin)来处理乳腺癌细胞是，也观察到这些抑制现象，但是当过量表达Hsp27时能够逆转槲皮苷的抑制作用。敲除Hsp也会抑制上皮间质转换(epithelial-mesenchymal transition, EMT)特征，比如降低蛋白snail和vimentin的表达，增加E-cadherin的表达。再者敲除Hsp也会将降低核转运以及ALDH+ BCSCs中的NF-kappa B活性，这是由于Ikappa B alpha表达增加的缘故。通过敲除Ikappa B alpha激活NF-kappa B能够逆转Hsp27 siRNA在抑制ALDH+ cells方面的抑制效应。
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  ]# i( F! \  ]4 b这些表明Hsp27通过调节上皮间质转换和NF-kappa B活性来促进BCSCs的维持。因为将Hsp27作为作用靶标可能是乳腺癌治疗中的一个新策略。