Impact of cyclin E overexpression on Smad3 activity in breast cancer cell lines
Impact of cyclin E overexpression on Smad3 activity in breast cancer cell lines
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DOI：
10.4161/cc.9.24.14158
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发表时间：
2010-12-15
期刊：
影响因子：
4.3
通讯作者：
Jeruss, Jacqueline S.
中科院分区：
文献类型：
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作者：
Cooley, Anne;Zelivianski, Stanislav;Jeruss, Jacqueline S.
Smad3是TGF β信号通路的一个组成部分，在乳腺癌细胞中参与G(1)阻滞。细胞周期有丝分裂原cyclin E的过度表达与乳腺癌预后不良有关，并且cyclin E/CDK2介导的Smad3磷酸化与Smad3活性抑制有关。我们假设过表达cyclin E的乳腺癌细胞的生物侵袭性可能与CDK2磷酸化和抑制Smad3的抑瘤作用有关。将含有空载体、野生型（WT） Smad3或CDK磷酸化位点突变的Smad3的表达构建物与Smad3应答报告构建物共转染到亲本、载体对照（A1）或过表达cyclin E （EL1）的MCF7细胞中。通过荧光素酶报告基因试验和mRNA分析评估Smad3的功能。我们还评估了Cdk2抑制剂和Cdk2 siRNA对Smad3活性的影响。与对照组或表达WT Smad3的细胞相比，含有CDK磷酸化位点突变的表达Smad3的细胞具有更高的p15和p21和更低的c-myc mRNA水平，以及更高的Smad3应答报告活性。与对照siRNA相比，转染cdk2 siRNA导致smad3应答报告基因活性显著增加；用Cdk2抑制剂治疗后，报告细胞活性也增加。因此，细胞周期蛋白e介导的Smad3抑制是由MCF7细胞中Smad3蛋白的CDK2磷酸化调控的。抑制CDK2可能导致乳腺癌细胞中Smad3肿瘤抑制因子活性的恢复，并且可能代表了一种治疗周期蛋白E过表达乳腺癌的潜在方法。
Smad3, a component of the TGF beta signaling pathway, contributes to G(1) arrest in breast cancer cells. Overexpression of the cell cycle mitogen, cyclin E, is associated with poor prognosis in breast cancer, and cyclin E/CDK2 mediated phosphorylation of Smad3 has been linked with inhibition of Smad3 activity. We hypothesized that the biological aggressiveness of cyclin E overexpressing breast cancer cells would be associated with CDK2 phosphorylation and inhibition of the tumor suppressant action of Smad3. Expression constructs containing empty vector, wild type (WT) Smad3 or Smad3 with CDK phosphorylation site mutations were co-transfected with a Smad3-responsive reporter construct into parental, vector control (A1) or cyclin E overexpressing (EL1) MCF7 cells. Smad3 function was evaluated by luciferase reporter assay and mRNA analysis. The impact of a Cdk2 inhibitor and cdk2 siRNA on Smad3 activity was also assessed. Cells expressing Smad3 containing mutations of the CDK phosphorylation sites had higher p15 and p21 and lower c-myc mRNA levels, as well as higher Smad3-responsive reporter activity, compared with controls or cells expressing WT Smad3. Transfection of cdk2 siRNA resulted in a significant increase in Smad3-responsive reporter activity compared with control siRNA; reporter activity was also increased after the treatment with a Cdk2 inhibitor. Thus, cyclin E-mediated inhibition of Smad3 is regulated by CDK2 phosphorylation of the Smad3 protein in MCF7 cells. Inhibition of CDK2 may lead to restoration of Smad3 tumor suppressor activity in breast cancer cells, and may represent a potential treatment approach for cyclin E overexpressing breast cancers.