Breast Department, International Peace Maternity and Child Health Hospital, Shanghai Jiaotong University, Shanghai, People's Republic of China.
Cell Oncol (Dordr). 2013 Feb;36(1):79-93. doi: 10.1007/s13402-012-0115-3. Epub 2012 Nov 27.
The signal transducer and activator of transcription 6 (Stat6), a member of the family of DNA-binding proteins, has been identified as a critical cell differentiation modulator in breast cancer cells. As of yet, the mechanisms underlying this function have remained largely unknown. To further elucidate the role of Stat6 in breast cancer development, we investigated the consequences of exogenous Stat6 expression.
Proliferation assays and flow cytometry assays were conducted to evaluate the putative role of Stat6 on cell proliferation. To this end, we produced synchronized cells after a double thymidine block, as confirmed by FACS analysis. mRNA levels of Stat6 were measured by RNase protection analysis. To confirm the interaction among proteins, we employed GST pull-down assays and immunoprecipitation assays. Luciferase assays and ChIP assays were used to assess the transcriptional activity.
Compared to control breast cancer cells, we found that exogenous Stat6 expression plays a critical role in controlling cell proliferation. Also in different breast tumor cell lines, endogenous Stat6 expression was found to be positively related to a lower proliferation rate. Interestingly, in human breast cancer cells Stat6 functions in G1/S cell cycle progression, and the growth-inhibitory effect of Stat6 was shown to be mediated by induction of the G1 cyclin-dependent kinase inhibitors p21(Cip1/WAF1) (p21) and p27(Kip1) (p27). Simultaneously, G1-related cyclin/cyclin-dependent kinase activities and pRB phosphorylation were markedly reduced, and cell cycle progression was blocked in the G1 phase. Stat6 knockdown resulted in enhanced cell proliferation and a decrease in p21 and p27 mRNA levels in the steroid-responsive and non-responsive T-47D and MDA-MB-231 cell lines, respectively. In addition, the stimulatory effect of Stat6 on p21 and p27 gene transcription was found to be associated with interaction of Stat6 with the transcription factor Sp1 at the proximal Sp1-binding sites in their respective promoters.
Together, these results identify Stat6 as an important cell differentiation regulatory protein functioning, at least in part, by interacting with Sp1 to activate the p21 and p27 gene promoters in breast cancer cells.
信号转导子和转录激活子 6（Stat6）是 DNA 结合蛋白家族的成员之一，已被鉴定为乳腺癌细胞中关键的细胞分化调节剂。然而，其功能的潜在机制在很大程度上仍然未知。为了进一步阐明 Stat6 在乳腺癌发展中的作用，我们研究了外源性 Stat6 表达的后果。
通过增殖测定和流式细胞术测定来评估 Stat6 对细胞增殖的潜在作用。为此，我们在双胸腺嘧啶阻断后产生了同步细胞，这一点通过 FACS 分析得到了证实。通过 RNase 保护分析测量 Stat6 的 mRNA 水平。为了确认蛋白质之间的相互作用，我们采用 GST 下拉测定和免疫沉淀测定。使用荧光素酶测定和 ChIP 测定来评估转录活性。
与对照乳腺癌细胞相比，我们发现外源性 Stat6 表达在控制细胞增殖方面起着关键作用。此外，在不同的乳腺癌肿瘤细胞系中，内源性 Stat6 表达与较低的增殖率呈正相关。有趣的是，在人类乳腺癌细胞中，Stat6 作用于 G1/S 细胞周期进程，Stat6 的生长抑制作用被证明是通过诱导 G1 周期蛋白依赖性激酶抑制剂 p21(Cip1/WAF1)（p21）和 p27(Kip1)（p27）来介导的。同时，G1 相关的周期蛋白/周期蛋白依赖性激酶活性和 pRB 磷酸化明显降低，细胞周期在 G1 期被阻断。Stat6 敲低导致类固醇反应性和非反应性 T-47D 和 MDA-MB-231 细胞系中的细胞增殖增强，以及 p21 和 p27 mRNA 水平降低。此外，发现 Stat6 对 p21 和 p27 基因转录的刺激作用与 Stat6 与转录因子 Sp1 之间的相互作用有关，该相互作用发生在它们各自启动子的近端 Sp1 结合位点处。
总之，这些结果表明 Stat6 是一种重要的细胞分化调节蛋白，通过与 Sp1 相互作用激活乳腺癌细胞中的 p21 和 p27 基因启动子，至少部分地发挥作用。
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