Hsp27 participates in the maintenance of breast cancer stem cells through regulation of epithelial-mesenchymal transition and nuclear factor-κB.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Department of Neurosurgery, Taipei Medical University-Shuang Ho Hospital, Jhongjheng Rd., No.291, New Taipei City, 23561, Taiwan.
Li
L
Wei
Tsung-Ta
TT
Liu
Hsiu-Huan
HH
Wang
Hui-Mei
HM
Hong
Alice L
AL
Yu
Hsiang-Pu
HP
Feng
Wen-Wei
WW
Chang
D000444:Aldehyde Dehydrogenase / Q000378:metabolism; D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor / Q000187:drug effects; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D005260:Female; D055551:HSP27 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D018832:Molecular Chaperones; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D014411:Neoplastic Stem Cells / Q000378:metabolism* / Q000473:pathology*; D010766:Phosphorylation; D011794:Quercetin / Q000494:pharmacology; D034741:RNA, Small Interfering; D023041:Xenograft Model Antitumor Assays
D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; C486699:Hspb2 protein, mouse; D018832:Molecular Chaperones; D016328:NF-kappa B; D034741:RNA, Small Interfering; D011794:Quercetin; D000444:Aldehyde Dehydrogenase; D051550:I-kappa B Kinase; D020928:Mitogen-Activated Protein Kinases