Wei Min, He Qi, Yang Zhongyin, Wang Zhiwei, Zhang Qing, Liu Bingya, Gu Qinlong, Su Liping, Yu Yingyan, Zhu Zhenggang, Zhang Guofeng
Breast Department, International Peace Maternity and Child Health Hospital, Shanghai Jiaotong University, Shanghai 200030, People's Republic of China.
BMC Cancer. 2014 Jan 8;14:10. doi: 10.1186/1471-2407-14-10.
Progesterone is essential for the proliferation and differentiation of mammary gland epithelium. Studies of breast cancer cells have demonstrated a biphasic progesterone response consisting of an initial proliferative burst followed by sustained growth arrest. However, the transcriptional factors acting with the progesterone receptor (PR) to mediate the effects of progesterone on mammary cell growth and differentiation remain to be determined. Recently, it was demonstrated that signal transducer and activator of transcription 6 (Stat6) is a cell growth suppressor. Similar to progesterone-bound PR, Stat6 acts by inducing the expression of the G1 cyclin-dependent kinase inhibitors p21 and p27. The possible interaction between Stat6 and progesterone pathways in mammary cells was therefore investigated in the present study.
ChIP and luciferase were assayed to determine whether Stat6 induces p21 and p27 expression by recruitment at the proximal Sp1-binding sites of the gene promoters. Immunoprecipitation and Western blotting were performed to investigate the interaction between Stat6 and PR-B. The cellular DNA content and cell cycle distribution in breast cancer cells were analyzed by FACS.
We found that Stat6 interacts with progesterone-activated PR in T47D cells. Stat6 synergizes with progesterone-bound PR to transactivate the p21 and p27 gene promoters at the proximal Sp1-binding sites. Moreover, Stat6 overexpression and knockdown, respectively, increased or prevented the induction of p21 and p27 gene expression by progesterone. Stat6 knockdown also abolished the inhibitory effects of progesterone on pRB phosphorylation, G1/S cell cycle progression, and cell proliferation. In addition, knockdown of Stat6 expression prevented the induction of breast cell differentiation markers, previously identified as progesterone target genes. Finally, Stat6 gene expression levels increased following progesterone treatment, indicating a positive auto-regulatory loop between PR and Stat6.
Taken together, these data identify Stat6 as a coactivator of PR mediating the growth-inhibitory and differentiation effects of progesterone on breast cancer cells.
孕酮对于乳腺上皮细胞的增殖和分化至关重要。对乳腺癌细胞的研究表明，孕酮反应呈双相性，最初是增殖爆发，随后是持续的生长停滞。然而，与孕酮受体（PR）共同作用以介导孕酮对乳腺细胞生长和分化影响的转录因子仍有待确定。最近，有研究表明信号转导子和转录激活子6（Stat6）是一种细胞生长抑制因子。与结合孕酮的PR类似，Stat6通过诱导G1期细胞周期蛋白依赖性激酶抑制剂p21和p27的表达发挥作用。因此，本研究对Stat6与乳腺细胞中孕酮信号通路之间可能的相互作用进行了研究。
采用染色质免疫沉淀法（ChIP）和荧光素酶检测法，以确定Stat6是否通过募集到基因启动子近端的Sp1结合位点来诱导p21和p27的表达。进行免疫沉淀和蛋白质印迹分析，以研究Stat6与PR-B之间的相互作用。通过流式细胞术分析乳腺癌细胞中的细胞DNA含量和细胞周期分布。
我们发现Stat6在T47D细胞中与孕酮激活的PR相互作用。Stat6与结合孕酮的PR协同作用，在近端Sp1结合位点反式激活p21和p27基因启动子。此外，Stat6的过表达和敲低分别增加或阻止了孕酮对p21和p27基因表达的诱导。Stat6敲低还消除了孕酮对pRB磷酸化、G1/S期细胞周期进程和细胞增殖的抑制作用。此外，Stat6表达的敲低阻止了先前被确定为孕酮靶基因的乳腺细胞分化标志物的诱导。最后，孕酮处理后Stat6基因表达水平升高，表明PR与Stat6之间存在正反馈调节环。
综上所述，这些数据表明Stat6是PR的共激活因子，介导孕酮对乳腺癌细胞的生长抑制和分化作用。
