Ku 蛋白与激活蛋白-2 转录因子相互作用，有助于乳腺癌细胞系中 ERBB2 的过表达。
Ku proteins interact with activator protein-2 transcription factors and contribute to ERBB2 overexpression in breast cancer cell lines.
机构信息
Laboratory of Molecular Oncology, GIGA Cancer, University of Liège, B34, avenue de l'hopital, Liege, 4000, Belgium.
出版信息
Breast Cancer Res. 2009;11(6):R83. doi: 10.1186/bcr2450. Epub 2009 Nov 11.
INTRODUCTION
Activator protein-2 (AP-2) alpha and AP-2gamma transcription factors contribute to ERBB2 gene overexpression in breast cancer. In order to understand the mechanism by which the ERBB2 gene is overexpressed we searched for novel AP-2 interacting factors that contribute to its activity.
METHODS
Ku proteins were identified as AP-2alpha interacting proteins by glutathione serine transferase (GST)-pull down followed by mass spectrometry. Transfection of the cells with siRNA, expression vectors and reporter vectors as well as chromatin immunoprecipitation (ChIP) assay were used to ascertain the implication of Ku proteins on ERBB2 expression.
RESULTS
Nuclear proteins from BT-474 cells overexpressing AP-2alpha and AP-2gamma were incubated with GST-AP2 or GST coated beads. Among the proteins retained specifically on GST-AP2 coated beads Ku70 and Ku80 proteins were identified by mass spectrometry. The contribution of Ku proteins to ERBB2 gene expression in BT-474 and SKBR3 cell lines was investigated by downregulating Ku proteins through the use of specific siRNAs. Depletion of Ku proteins led to downregulation of ERBB2 mRNA and protein levels. Furthermore, reduction of Ku80 in HCT116 cell line decreased the AP-2alpha activity on a reporter vector containing an AP-2 binding site linked to the ERBB2 core promoter, and transfection of Ku80 increased the activity of AP-2alpha on this promoter. Ku siRNAs also inhibited the activity of this reporter vector in BT-474 and SKBR3 cell lines and the activity of the ERBB2 promoter was further reduced by combining Ku siRNAs with AP-2alpha and AP-2gamma siRNAs. ChIP experiments with chromatin extracted from wild type or AP-2alpha and AP-2gamma or Ku70 siRNA transfected BT-474 cells demonstrated Ku70 recruitment to the ERBB2 proximal promoter in association with AP-2alpha and AP-2gamma. Moreover, Ku70 siRNA like AP-2 siRNAs, greatly reduced PolII recruitment to the ERBB2 proximal promoter.
CONCLUSIONS
Ku proteins in interaction with AP-2 (alpha and gamma) contribute to increased ERBB2 mRNA and protein levels in breast cancer cells.
简介
激活蛋白-2（AP-2）alpha 和 AP-2gamma 转录因子有助于乳腺癌中 ERBB2 基因的过度表达。为了了解 ERBB2 基因过度表达的机制，我们寻找了新的与 AP-2 相互作用的因子，这些因子有助于其活性。
方法
通过谷胱甘肽 S-转移酶（GST）下拉结合质谱鉴定 Ku 蛋白为 AP-2alpha 相互作用蛋白。通过转染 siRNA、表达载体和报告载体以及染色质免疫沉淀（ChIP）实验，确定 Ku 蛋白对 ERBB2 表达的影响。
结果
用 GST-AP2 或 GST 包被珠孵育过表达 AP-2alpha 和 AP-2gamma 的 BT-474 细胞的核蛋白。通过质谱鉴定，在 GST-AP2 包被珠上特异性保留的蛋白质中，鉴定出 Ku70 和 Ku80 蛋白。通过使用特异性 siRNA 下调 Ku 蛋白，研究 Ku 蛋白在 BT-474 和 SKBR3 细胞系中对 ERBB2 基因表达的贡献。Ku 蛋白的耗竭导致 ERBB2 mRNA 和蛋白水平下调。此外，在 HCT116 细胞系中减少 Ku80 降低了与 ERBB2 核心启动子相连的包含 AP-2 结合位点的报告载体上的 AP-2alpha 活性，并且 Ku80 的转染增加了该启动子上的 AP-2alpha 活性。Ku siRNAs 也抑制了 BT-474 和 SKBR3 细胞系中该报告载体的活性，并且结合 Ku siRNAs 与 AP-2alpha 和 AP-2gamma siRNAs 进一步降低了 ERBB2 启动子的活性。用来自野生型或 AP-2alpha 和 AP-2gamma 或 Ku70 siRNA 转染的 BT-474 细胞提取的染色质进行 ChIP 实验表明，Ku70 与 AP-2alpha 和 AP-2gamma 一起募集到 ERBB2 近端启动子。此外，像 AP-2 siRNAs 一样，Ku70 siRNA 大大减少了 PolII 募集到 ERBB2 近端启动子。
结论
与 AP-2（alpha 和 gamma）相互作用的 Ku 蛋白有助于增加乳腺癌细胞中 ERBB2 mRNA 和蛋白水平。
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