Egger Jacklynn V, Lane Maria V, Antonucci Lisa A, Dedi Brixhilda, Krucher Nancy A
a Department of Biology , Dyson Hall, Pace University , Pleasantville , NY , USA.
Cancer Biol Ther. 2016 Nov;17(11):1197-1205. doi: 10.1080/15384047.2016.1235668. Epub 2016 Sep 19.
The tumor suppressor Retinoblastoma (Rb) protein is highly phosphorylated in cancer cells largely due to the overexpression of cyclins or the loss of expression of cyclin dependent kinase inhibitors (cdki). Hyperphosphorylation of Rb promotes proliferation, and plays a role in the regulation of apoptosis. Recently, inhibition of cyclin dependent activity toward Rb has been identified as a strategy that has shown clinical efficacy. We utilized a method to induce phosphatase activity toward Rb in cells by shRNA silencing of PNUTS (Phosphatase Nuclear Targeting Subunit) that regulates PP1-mediated dephosphorylation of Rb. In this study, the effect of Rb dephosphorylation on the epithelial to mesenchymal transition (EMT) was determined. The EMT transition is observed in cancer cells that have acquired invasive characteristics. In breast cancer cells grown in 3D Matrigel cultures, MCF7 cells undergo apoptosis in response to Rb dephosphorylation, whereas MDA-MB-231 and Hs578T cells exhibit a reduction in the EMT. Cells devoid of phosphorylated Rb (nontransformed MCF10A and Rb-null MDA-MB-468) lacked any response to PNUTS depletion, showing the effect is Rb-dependent. In addition, these studies showed that Rb dephosphorylation in 3D Matrigel cultures of highly invasive HT1080 cells led to the inhibition of the EMT. Furthermore we observed association between dephosphorylated Rb with ZEB1, a zinc-finger E-box-binding transcription factor that regulates expression of E- and N-cadherins. Finally Rb dephosphorylation led to inhibition of ZEB1 transcriptional activity, this data supports the notion that Rb dephosphorylation modulates the EMT. These studies suggest targeting Rb phosphorylation in mesenchymal cancer cells may decrease invasiveness.
肿瘤抑制蛋白视网膜母细胞瘤（Rb）在癌细胞中高度磷酸化，这主要归因于细胞周期蛋白的过表达或细胞周期蛋白依赖性激酶抑制剂（cdki）表达的缺失。Rb的过度磷酸化促进细胞增殖，并在细胞凋亡调控中发挥作用。最近，抑制细胞周期蛋白对Rb的依赖性活性已被确定为一种具有临床疗效的策略。我们利用一种方法，通过对调节PP1介导的Rb去磷酸化的PNUTS（磷酸酶核靶向亚基）进行shRNA沉默，在细胞中诱导针对Rb的磷酸酶活性。在本研究中，确定了Rb去磷酸化对上皮-间质转化（EMT）的影响。EMT转变在具有侵袭特性的癌细胞中可见。在3D基质胶培养的乳腺癌细胞中，MCF7细胞因Rb去磷酸化而发生凋亡，而MDA-MB-231和Hs578T细胞的EMT则有所减少。缺乏磷酸化Rb的细胞（未转化的MCF10A和Rb基因缺失的MDA-MB-468）对PNUTS缺失无任何反应，表明该效应依赖于Rb。此外，这些研究表明，在高侵袭性HT1080细胞的3D基质胶培养中，Rb去磷酸化导致EMT受到抑制。此外，我们观察到去磷酸化的Rb与ZEB1之间存在关联，ZEB1是一种锌指E盒结合转录因子，可调节E-钙黏蛋白和N-钙黏蛋白的表达。最后，Rb去磷酸化导致ZEB1转录活性受到抑制，这一数据支持Rb去磷酸化调节EMT这一观点。这些研究表明，针对间充质癌细胞中的Rb磷酸化进行靶向治疗可能会降低其侵袭性。
