Alqahtani H, Gopal K, Gupta N, Jung K, Alshareef A, Ye X, Wu F, Li L, Lai R
Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada.
Department of Oncology, University of Alberta, Edmonton, Alberta, Canada.
Cell Signal. 2016 Feb;28(2):42-50. doi: 10.1016/j.cellsig.2015.11.004. Epub 2015 Nov 10.
We have previously demonstrated the existence of two phenotypically distinct cell subsets in estrogen receptor (ER)-positive breast cancer (BC) based on their differential response to a Sox2 reporter (SRR2), with reporter responsive (RR) cells being more tumorigenic and stem-like than reporter unresponsive (RU) cells. To delineate the molecular mechanisms underlying this phenotypic dichotomy, we tested our hypothesis that Sox2, which is a key regulator of the RR phenotype, is under the control of its binding partners. In this study, we focused on DDX17, known to be a transcription co-activator and found to be a Sox2 binding partner by liquid chromatography-mass spectrometry. Using immunoprecipitation, we confirmed the binding between DDX17 and Sox2, although this interaction was largely restricted to RR cells. While DDX17 was found in both the cytoplasm and nuclei in RU cells, it is confined to the nuclei in RR cells. siRNA knockdown of DDX17 in RR cells substantially decreased the Sox2-SRR2 binding and significantly decreased the SRR2 reporter activity without affecting the protein level of Sox2. Using ChIP-PCR, DDX17 knockdown also significantly decreased the binding of Sox2 to genomic SRR2, as well as 3 of its specific gene targets including MUC15, CCND1 and CD133. Correlating with these findings, siRNA knockdown of DDX17 significantly reduced soft agar colony formation and mammosphere formation in RR cells but not RU cells. To conclude, DDX17 is a Sox2-binding protein in ER-positive BC. In RR but not RU cells, DDX17 enhances the tumorigenic and stem-like features of Sox2 by promoting its binding to its target genes.
我们之前已经证明，基于雌激素受体（ER）阳性乳腺癌（BC）细胞对Sox2报告基因（SRR2）的不同反应，存在两种表型不同的细胞亚群，其中报告基因反应性（RR）细胞比报告基因无反应性（RU）细胞更具致瘤性且更具干细胞样特性。为了阐明这种表型二分法背后的分子机制，我们检验了我们的假设，即作为RR表型关键调节因子的Sox2受其结合伙伴的调控。在本研究中，我们聚焦于已知为转录共激活因子且通过液相色谱 - 质谱法发现是Sox2结合伙伴的DDX17。通过免疫沉淀，我们证实了DDX17与Sox2之间的结合，尽管这种相互作用主要局限于RR细胞。虽然在RU细胞的细胞质和细胞核中都发现了DDX17，但它在RR细胞中局限于细胞核。在RR细胞中通过siRNA敲低DDX17可显著降低Sox2 - SRR2的结合，并显著降低SRR2报告基因活性，而不影响Sox2的蛋白水平。使用染色质免疫沉淀 - 聚合酶链反应（ChIP - PCR），敲低DDX17也显著降低了Sox2与基因组SRR2及其3个特定基因靶点（包括MUC15、CCND1和CD133）的结合。与这些发现相关的是，在RR细胞而非RU细胞中，通过siRNA敲低DDX17显著减少了软琼脂集落形成和乳腺球形成。总之，DDX17是ER阳性BC中的一种Sox2结合蛋白。在RR细胞而非RU细胞中，DDX17通过促进Sox2与其靶基因的结合来增强其致瘤性和干细胞样特性。
